Processes and intermediates for making a JAK inhibitor

ABSTRACT

This invention relates to processes and intermediates for making {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, useful in the treatment of diseases related to the activity of Janus kinases (JAK) including inflammatory disorders, autoimmune disorders, cancer, and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/645,046, filed Mar. 11, 2015, which is a divisional of U.S.application Ser. No. 14/197,701, filed Mar. 5, 2014, which claims thebenefit of U.S. Provisional Appl. No. 61/773,659, filed Mar. 6, 2013,each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to processes and intermediates for making{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,useful in the treatment of diseases related to the activity of Januskinases (JAK) including inflammatory disorders, autoimmune disorders,cancer, and other diseases.

BACKGROUND

Protein kinases (PKs) regulate diverse biological processes includingcell growth, survival, differentiation, organ formation, morphogenesis,neovascularization, tissue repair, and regeneration, among others.Protein kinases also play specialized roles in a host of human diseasesincluding cancer. Cytokines, low-molecular weight polypeptides orglycoproteins, regulate many pathways involved in the host inflammatoryresponse to sepsis. Cytokines influence cell differentiation,proliferation and activation, and can modulate both pro-inflammatory andanti-inflammatory responses to allow the host to react appropriately topathogens. Signaling of a wide range of cytokines involves the Januskinase family (JAKs) of protein tyrosine kinases and Signal Transducersand Activators of Transcription (STATs). There are four known mammalianJAKs: JAK1 (Janus kinase-1), JAK2, JAK3 (also known as Janus kinase,leukocyte; JAKL; and L-JAK), and TYK2 (protein-tyrosine kinase 2).

Cytokine-stimulated immune and inflammatory responses contribute topathogenesis of diseases: pathologies such as severe combinedimmunodeficiency (SCID) arise from suppression of the immune system,while a hyperactive or inappropriate immune/inflammatory responsecontributes to the pathology of autoimmune diseases (e.g., asthma,systemic lupus erythematosus, thyroiditis, myocarditis), and illnessessuch as scleroderma and osteoarthritis (Ortmann, R. A., T. Cheng, et al.(2000) Arthritis Res 2(1): 16-32).

Deficiencies in expression of JAKs are associated with many diseasestates. For example, Jak1−/− mice are runted at birth, fail to nurse,and die perinatally (Rodig, S. J., M. A. Meraz, et al. (1998) Cell93(3): 373-83). Jak2−/− mouse embryos are anemic and die around day 12.5postcoitum due to the absence of definitive erythropoiesis.

The JAK/STAT pathway, and in particular all four JAKs, are believed toplay a role in the pathogenesis of asthmatic response, chronicobstructive pulmonary disease, bronchitis, and other relatedinflammatory diseases of the lower respiratory tract. Multiple cytokinesthat signal through JAKs have been linked to inflammatorydiseases/conditions of the upper respiratory tract, such as thoseaffecting the nose and sinuses (e.g., rhinitis and sinusitis) whetherclassically allergic reactions or not. The JAK/STAT pathway has alsobeen implicated in inflammatory diseases/conditions of the eye andchronic allergic responses.

Activation of JAK/STAT in cancers may occur by cytokine stimulation(e.g. IL-6 or GM-CSF) or by a reduction in the endogenous suppressors ofJAK signaling such as SOCS (suppressor or cytokine signaling) or PIAS(protein inhibitor of activated STAT) (Boudny, V., and Kovarik, J.,Neoplasm. 49:349-355, 2002). Activation of STAT signaling, as well asother pathways downstream of JAKs (e.g., Akt), has been correlated withpoor prognosis in many cancer types (Bowman, T., et al. Oncogene19:2474-2488, 2000). Elevated levels of circulating cytokines thatsignal through JAK/STAT play a causal role in cachexia and/or chronicfatigue. As such, JAK inhibition may be beneficial to cancer patientsfor reasons that extend beyond potential anti-tumor activity.

JAK2 tyrosine kinase can be beneficial for patients withmyeloproliferative disorders, e.g., polycythemia vera (PV), essentialthrombocythemia (ET), myeloid metaplasia with myelofibrosis (MMM)(Levin, et al., Cancer Cell, vol. 7, 2005: 387-397). Inhibition of theJAK2V617F kinase decreases proliferation of hematopoietic cells,suggesting JAK2 as a potential target for pharmacologic inhibition inpatients with PV, ET, and MMM.

Inhibition of the JAKs may benefit patients suffering from skin immunedisorders such as psoriasis, and skin sensitization. The maintenance ofpsoriasis is believed to depend on a number of inflammatory cytokines inaddition to various chemokines and growth factors (JCI, 113:1664-1675),many of which signal through JAKs (Adv Pharmacol. 2000; 47:113-74).

JAK1 plays a central role in a number of cytokine and growth factorsignaling pathways that, when dysregulated, can result in or contributeto disease states. For example, IL-6 levels are elevated in rheumatoidarthritis, a disease in which it has been suggested to have detrimentaleffects (Fonesca, J. E. et al., Autoimmunity Reviews, 8:538-42, 2009).Because IL-6 signals, at least in part, through JAK1, antagonizing IL-6directly or indirectly through JAK1 inhibition is expected to provideclinical benefit (Guschin, D., N., et al Embo J 14:1421, 1995; Smolen,J. S., et al. Lancet 371:987, 2008). Moreover, in some cancers JAK1 ismutated resulting in constitutive undesirable tumor cell growth andsurvival (Mullighan C G, Proc Natl Acad Sci USA. 106:9414-8, 2009; FlexE., et al. J Exp Med. 205:751-8, 2008). In other autoimmune diseases andcancers elevated systemic levels of inflammatory cytokines that activateJAK1 may also contribute to the disease and/or associated symptoms.Therefore, patients with such diseases may benefit from JAK1 inhibition.Selective inhibitors of JAK1 may be efficacious while avoidingunnecessary and potentially undesirable effects of inhibiting other JAKkinases.

Selective inhibitors of JAK1, relative to other JAK kinases, may havemultiple therapeutic advantages over less selective inhibitors. Withrespect to selectivity against JAK2, a number of important cytokines andgrowth factors signal through JAK2 including, for example,erythropoietin (Epo) and thrombopoietin (Tpo) (Parganas E, et al. Cell.93:385-95, 1998). Epo is a key growth factor for red blood cellsproduction; hence a paucity of Epo-dependent signaling can result inreduced numbers of red blood cells and anemia (Kaushansky K, NEJM354:2034-45, 2006). Tpo, another example of a JAK2-dependent growthfactor, plays a central role in controlling the proliferation andmaturation of megakaryocytes—the cells from which platelets are produced(Kaushansky K, NEJM 354:2034-45, 2006). As such, reduced Tpo signalingwould decrease megakaryocyte numbers (megakaryocytopenia) and lowercirculating platelet counts (thrombocytopenia). This can result inundesirable and/or uncontrollable bleeding. Reduced inhibition of otherJAKs, such as JAK3 and Tyk2, may also be desirable as humans lackingfunctional version of these kinases have been shown to suffer fromnumerous maladies such as severe-combined immunodeficiency orhyperimmunoglobulin E syndrome (Minegishi, Y, et al. Immunity 25:745-55,2006; Macchi P, et al. Nature. 377:65-8, 1995). Therefore a JAK1inhibitor with reduced affinity for other JAKs would have significantadvantages over a less-selective inhibitor with respect to reduced sideeffects involving immune suppression, anemia and thrombocytopenia.

Due to the usefulness of JAK inhibitors, there is a need for developmentof new processes for making JAK inhibitors. This invention is directedtowards this need and others.

SUMMARY

JAK inhibitors are described in US 2011/0224190, which is incorporatedherein by reference in its entirety, including{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,which is depicted below as Formula I.

The present invention provides, inter alia, processes and intermediatesfor making the compound of Formula I. In particular, the presentinvention provides processes of making a compound of Formula II:

comprising reacting a compound of Formula III:

with a compound of Formula IV:

under Suzuki coupling conditions to form a compound of Formula II,wherein:

Z is H or a protecting group;

P¹ is a protecting group;

X¹ is halo; and

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

The present invention further provides processes for making a compoundof Formula IIa:

comprising reacting a compound of Formula IIIa:

with a compound of Formula IVa:

under Suzuki coupling conditions to form a compound of Formula IIa,wherein the Suzuki coupling conditions comprise heating a reactionmixture comprising the compound of Formula IIIa, the compound of FormulaIVa, [1,1′-bis(dicyclohexylphosphino)ferrocene]dichloropalladium (II),cesium fluoride, and a solvent component, wherein the solvent componentcomprises water and tert-butanol.

The process further comprises a process for deprotecting a compound ofFormula II or IIa to form a compound of Formula V:

or salt thereof.

The present invention also provides a process further comprisingreacting a compound of Formula V, or a salt thereof, with a compound ofFormula VI:

in the presence of a reducing agent to form a compound of Formula I:

or a salt thereof.

The present invention further provides compounds of Formula VII:

or salts thereof; wherein:

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

The present invention further provides processes for making a compoundof Formula VII, comprising reacting a compound of Formula VIII:

with a compound of Formula IX:

in the presence of a coupling agent to form a compound of Formula VII;wherein:

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

The present invention further provides processes of making a compound ofFormula VIIa, comprising reacting a compound of Formula VIII, or a saltthereof:

with a compound of Formula IXa:

in the presence of a coupling agent to form a compound of Formula VIIa:

The present invention further provides processes for making a compoundof Formula I, comprising reacting the compound of Formula VII or VIIawith a compound of Formula IVa:

under Suzuki coupling conditions to form a compound of Formula I:

wherein the Suzuki coupling conditions comprise heating a reactionmixture comprising the compound of Formula VII or VIIa, the compound ofFormula IVa, a Suzuki coupling catalyst, a base and a solvent component.

The present invention further provides a compound of Formula VIII:

or a salt thereof.

The present invention further provides processes of preparing a compoundof Formula VIII, or a salt thereof, comprising reacting a compound ofFormula VI:

with a compound of Formula X:

or a salt thereof, in the presence of a reducing agent.

The present invention further provides processes of preparing a compoundof Formula III, comprising reacting a compound of Formula X:

or salt thereof, with a compound of Formula IX:

in the presence of a coupling agent to form a compound of Formula III,or salt thereof; wherein:

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

DETAILED DESCRIPTION

At various places in the present specification, substituents ofcompounds of the invention are disclosed in groups or in ranges. It isspecifically intended that the invention include each and everyindividual subcombination of the members of such groups and ranges. Forexample, the term “C₁₋₆ alkyl” is specifically intended to individuallydisclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment. Conversely,various features of the invention which are, for brevity, described inthe context of a single embodiment, can also be provided separately orin any suitable subcombination.

The term “n-membered” where n is an integer typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, piperidinyl is an example of a6-membered heterocycloalkyl ring and 1,2,3,4-tetrahydro-naphthalene isan example of a 10-membered cycloalkyl group.

For compounds of the invention in which a variable appears more thanonce, each variable can be a different moiety independently selectedfrom the group defining the variable. For example, where a structure isdescribed having two R groups that are simultaneously present on thesame compound, the two R groups can represent different moietiesindependently selected from the group defined for R.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is understoodthat substitution at a given atom is limited by valency.

As used herein, the term “alkyl”, employed alone or in combination withother terms, refers to a saturated hydrocarbon group that may bestraight-chain or branched. In some embodiments, the alkyl groupcontains 1 to 12, 1 to 8, or 1 to 6 carbon atoms. Examples of alkylmoieties include, but are not limited to, chemical groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl,sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl,n-hexyl, 1,2,2-trimethylpropyl, n-heptyl, n-octyl, and the like. In someembodiments, the alkyl moiety is methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,or 2,4,4-trimethylpentyl. In some embodiments, the alkyl moiety ismethyl.

As used herein, the terms “halo” and “halogen”, employed alone or incombination with other terms, refer to fluoro, chloro, bromo, and iodo.In some embodiments, halo is chloro, bromo, or iodo. In someembodiments, halo is chloro.

As used herein, “heterocycloalkyl” refers to an non-aromatic monocyclicring including cyclized alkyl or alkenyl groups where one or more of thering-forming carbon atoms is replaced by a heteroatom such as an O, N,S, or B atom.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C), infrared spectroscopy, orspectrophotometry (e.g., UV-visible); or by chromatography such as highperformance liquid chromatograpy (HPLC) or thin layer chromatography(TLC) or other related techniques.

As used herein, the term “reacting” is used as known in the art andgenerally refers to the bringing together of chemical reagents in such amanner so as to allow their interaction at the molecular level toachieve a chemical or physical transformation. In some embodiments, thereacting involves two reagents, wherein one or more equivalents ofsecond reagent are used with respect to the first reagent. The reactingsteps of the processes described herein can be conducted for a time andunder conditions suitable for preparing the identified product.

Preparation of compounds can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in Greene, et al., Protective Groups in OrganicSynthesis, 4d. Ed., Wiley & Sons, 2007, which is incorporated herein byreference in its entirety. Adjustments to the protecting groups andformation and cleavage methods described herein may be adjusted asnecessary in light of the various substituents.

The reactions of the processes described herein can be carried out insuitable solvents which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected. In some embodiments, reactionscan be carried out in the absence of solvent, such as when at least oneof the reagents is a liquid or gas.

Suitable solvents can include halogenated solvents such as carbontetrachloride, bromodichloromethane, dibromochloromethane, bromoform,chloroform, bromochloromethane, dibromomethane, butyl chloride,dichloromethane, tetrachloroethylene, trichloroethylene,1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethane,2-chloropropane, α,α,α-trifluorotoluene, 1,2-dichloroethane,1,2-dibromoethane, hexafluorobenzene, 1,2,4-trichlorobenzene,1,2-dichlorobenzene, chlorobenzene, fluorobenzene, mixtures thereof andthe like.

Suitable ether solvents include: dimethoxymethane, tetrahydrofuran,1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethylether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether,diethylene glycol diethyl ether, triethylene glycol dimethyl ether,anisole, t-butyl methyl ether, mixtures thereof and the like.

Suitable protic solvents can include, by way of example and withoutlimitation, water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol,2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol,2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butylalcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, or 3-pentanol,neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethylether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol,phenol, or glycerol.

Suitable aprotic solvents can include, by way of example and withoutlimitation, tetrahydrofuran (THF), N,N-dimethylformamide (DMF),N,N-dimethylacetamide (DMA),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP),formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethylsulfoxide, propionitrile, ethyl formate, methyl acetate,hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate,sulfolane, N,N-dimethylpropionamide, tetramethylurea, nitromethane,nitrobenzene, or hexamethylphosphoramide.

Suitable hydrocarbon solvents include benzene, cyclohexane, pentane,hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene,m-, o-, or p-xylene, octane, indane, nonane, or naphthalene.

The reactions of the processes described herein can be carried out atappropriate temperatures which can be readily determined by the skilledartisan. Reaction temperatures will depend on, for example, the meltingand boiling points of the reagents and solvent, if present; thethermodynamics of the reaction (e.g., vigorously exothermic reactionsmay need to be carried out at reduced temperatures); and the kinetics ofthe reaction (e.g., a high activation energy barrier may need elevatedtemperatures). “Elevated temperature” refers to temperatures above roomtemperature (about 22° C.).

The reactions of the processes described herein can be carried out inair or under an inert atmosphere. Typically, reactions containingreagents or products that are substantially reactive with air can becarried out using air-sensitive synthetic techniques that are well knownto the skilled artisan.

In some embodiments, preparation of compounds can involve the additionof acids or bases to affect, for example, catalysis of a desiredreaction or formation of salt forms such as acid addition salts.

Example acids can be inorganic or organic acids. Inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, andnitric acid. Organic acids include formic acid, acetic acid, propionicacid, butanoic acid, benzoic acid, 4-nitrobenzoic acid, methanesulfonicacid, p-toluenesulfonic acid, benzenesulfonic acid, tartaric acid,trifluoroacetic acid, propiolic acid, butyric acid, 2-butynoic acid,vinyl acetic acid, pentanoic acid, hexanoic acid, heptanoic acid,octanoic acid, nonanoic acid and decanoic acid.

Example bases include lithium hydroxide, sodium hydroxide, potassiumhydroxide, lithium carbonate, sodium carbonate, potassium carbonate, andsodium bicarbonate. Some example strong bases include, but are notlimited to, hydroxide, alkoxides, metal amides, metal hydrides, metaldialkylamides and arylamines, wherein; alkoxides include lithium, sodiumand potassium salts of methyl, ethyl and t-butyl oxides; metal amidesinclude sodium amide, potassium amide and lithium amide; metal hydridesinclude sodium hydride, potassium hydride and lithium hydride; and metaldialkylamides include sodium and potassium salts of methyl, ethyl,n-propyl, i-propyl, n-butyl, t-butyl, trimethylsilyl and cyclohexylsubstituted amides.

The intermediates and products may also include salts of the compoundsdisclosed herein. As used herein, the term “salt” refers to a saltformed by the addition of an acceptable acid or base to a compounddisclosed herein. In some embodiments, the salts are pharmaceuticallyacceptable salts. As used herein, the phrase “pharmaceuticallyacceptable” refers to a substance that is acceptable for use inpharmaceutical applications from a toxicological perspective and doesnot adversely interact with the active ingredient. Pharmaceuticallyacceptable salts, including mono- and bi-salts, include, but are notlimited to, those derived from organic and inorganic acids such as, butnot limited to, acetic, lactic, citric, cinnamic, tartaric, succinic,fumaric, maleic, malonic, mandelic, malic, oxalic, propionic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, glycolic,pyruvic, methanesulfonic, ethanesulfonic, toluenesulfonic, salicylic,benzoic, and similarly known acceptable acids. Lists of suitable saltsare found in Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Company, Easton, Pa., 1985, p. 1418 and Journal ofPharmaceutical Science, 66, 2 (1977), each of which is incorporatedherein by reference in their entireties.

Upon carrying out preparation of compounds according to the processesdescribed herein, the usual isolation and purification operations suchas concentration, filtration, extraction, solid-phase extraction,recrystallization, chromatography, and the like may be used, to isolatethe desired products.

In some embodiments, the compounds described herein and salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compound of theinvention. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compound of the invention, or a saltthereof. Methods for isolating compounds and their salts are routine inthe art.

Processes for preparing some of the intermediates can be found in U.S.Provisional Patent Appl. No. 61/531,896, filed Sep. 7, 2011, U.S. patentapplication Ser. No. 12/687,623, filed Jan. 14, 2010, and U.S. patentapplication Ser. No. 13/043,986, filed Mar. 9, 2011, each of which isincorporated herein by reference in its entirety.

Processes and Intermediates

The present invention provides, inter alia, processes and intermediatesfor making the compound of Formula I. Accordingly, in one aspect, thepresent invention provides a process, comprising:

reacting a compound of Formula III:

with a compound of Formula IV:

under Suzuki coupling conditions to form a compound of Formula II:

wherein:

Z is H or a protecting group;

P¹ is a protecting group;

X¹ is halo; and

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

In some embodiments, P¹ is tert-butoxycarbonyl. Appropriate P₁protecting groups include, but are not limited to the protecting groupsfor amines delineated in Wuts and Greene, Protective Groups in OrganicSynthesis, 4th ed., John Wiley & Sons: New Jersey, pages 696-887 (and,in particular, pages 872-887) (2007), which is incorporated herein byreference in its entirety. In some embodiments, P₁ is benzyloxycarbonyl(Cbz), 2,2,2-trichloroethoxycarbonyl (Troc),2-(trimethylsilyl)ethoxycarbonyl (Teoc),2-(4-trifluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc),t-butoxycarbonyl (BOC), 1-adamantyloxycarbonyl (Adoc),2-adamantylcarbonyl (2-Adoc), 2,4-dimethylpent-3-yloxycarbonyl (Doc),cyclohexyloxycarbonyl (Hoc), 1,1-dimethyl-2,2,2-trichloroethoxycarbonyl(TcBOC), vinyl, 2-chloroethyl, 2-phenylsulfonylethyl, allyl, benzyl,2-nitrobenzyl, 4-nitrobenzyl, diphenyl-4-pyridylmethyl,N′,N′-dimethylhydrazinyl, methoxymethyl, t-butoxymethyl (Bum),benzyloxymethyl (BOM), or 2-tetrahydropyranyl (THP). In someembodiments, P₁ is tri(C₁₋₄alkyl)silyl (e.g., tri(isopropyl)silyl). Insome embodiments, P₁ is 1,1-diethoxymethyl. In some embodiments, P₁ is2-(trimethylsilyl)ethoxymethyl (SEM). In some embodiments, P₁ isN-pivaloyloxymethyl (POM).

In some embodiments,

In some embodiments, R¹ and R² are each independently methyl or ethyl.In some embodiments, R¹ and R² are each methyl. In some embodiments, R¹and R² are each ethyl.

In some embodiments, X¹ is chloro.

In some embodiments, Z is H.

In some embodiments, the compound of Formula III has Formula IIIa:

In some embodiments, the compound of Formula IV has Formula IVa:

In some embodiments, the Suzuki coupling conditions comprise heating areaction mixture comprising the compound of Formula III, the compound ofFormula IV, a Suzuki coupling catalyst, a base and a solvent component.

The Suzuki coupling reaction in the processes described herein can beinitiated using a number of different known Suzuki catalysts, includingpalladium(0) and palladium(II) catalysts and performed under conditionsknown in the art (see, e.g., Miyaura and Suzuki, Chem. Rev. 1995, 95,2457-2483, which is hereby incorporated in its entirety). In someembodiments, “in the presence of a catalyst” may refer to the additionof a catalyst precursor, which is present in some other form during thereaction cycle. In some embodiments, the palladium catalyst is Pd(PPh₃)₄and Pd(dppf)₂Cl₂. In some embodiments, the catalyst is[1,1′-bis(dicyclohexylphosphino)ferrocene]dichloropalladium (II). Insome embodiments, the palladium catalyst is[1,1′-bis(dicyclohexylphosphino)ferrocene]dichloropalladium (II)(“Pd-127”), tetrakis(triphenylphosphine)palladium(0), ortetrakis(tri(o-tolyl)phosphine)palladium(0). In some embodiments, thepalladium catalyst is tetrakis(triphenylphosphine) palladium(0). In someembodiments, the palladium catalyst loading is from about 1×10⁻⁴ toabout 0.1 equivalents. In some embodiments, the palladium catalystloading is from about 0.0010 to about 0.0015 equivalents.

In some embodiments, the base is cesium fluoride. In some embodiments,the cesium fluoride is present in 3 equivalents or more (e.g., 3.5equivalents) based on the compound of Formula IV. In some embodiments,the solvent component can include tert-butanol and water. In someembodiments, the tert-butanol and water are present in a 1:1 volumeratio.

In some embodiments, compounds of Formula III and IV are present inabout a 1:1 molar ratio.

In some embodiments, the solvent component comprises water and anorganic solvent. In some embodiments, the organic solvent is1,4-dioxane, 1-butanol, t-butanol, 1,2-dimethoxyethane (DME), DMF,2-propanol, toluene or ethanol, or a combination thereof.

In some embodiments, the base is an inorganic base. In some embodiments,the base is an organic base. In some embodiments, the base is an alkalimetal carbonate (e.g., K₂CO₃ or Na₂CO₃). In some embodiments, the baseis potassium carbonate (K₂CO₃) or CsF. In some embodiments, two to fiveequivalents of base (e.g., K₂CO₃, CsF) are used.

In some embodiments, the Suzuki coupling reaction is conducted at atemperature of about 80° C. to about 100° C. In some embodiments, thereaction is carried out for two to twelve hours. In some embodiments,the compound of Formula II or IIa can be optionally isolated fromaqueous work-up of the Suzuki coupling reaction mixture or directlyused.

In another aspect, the present invention provides processes for making acompound of Formula IIa, comprising reacting a compound of Formula IIIa:

with a compound of Formula IVa:

under Suzuki coupling conditions to form a compound of Formula IIa:

wherein the Suzuki coupling conditions comprise heating a reactionmixture comprising the compound of Formula IIIa, the compound of FormulaIVa, [1,1′-bis(dicyclohexylphosphino)ferrocene]dichloropalladium (II),cesium fluoride, and a solvent component, wherein the solvent componentcomprises water and tert-butanol.

The processes for making a compound of Formula II or IIa further cancomprise deprotecting the compound of Formula II to form a compound ofFormula V:

or salt thereof. The deprotecting can include reacting the compound ofFormula II or Formula IIa with hydrochloric acid (e.g., about 5 Mhydrochloric acid) in a second solvent component (e.g., water anddichloromethane). In some embodiments, the hydrochloric acid is used inan amount of 5 to 8 equivalents based on the compound of Formula II. Asused herein, “second” in the phrase “second solvent component” is usedto differentiate the solvent component from other solvent componentsused in earlier or later steps of the process and does not indicate thattwo solvents must be present.

In some embodiments, the compound of Formula V, or a salt thereof, isfurther reacted with a compound of Formula VI:

in the presence of a reducing agent to form a compound of Formula I:

or a salt thereof.

In some embodiments, the reducing agent is sodium cyanoborohydride orsodium triacetoxyborohydride. In some embodiments, the reducing agent issodium triacetoxyborohydride. In some embodiments, greater than 1equivalent (e.g., 2 equivalents) of sodium triacetoxyborohydride is usedbased on the compound of Formula V.

The reducing agent can be any reducing agent suitable for use inreductive amination, including various borohydride and borane reducingagents, such as those in Ellen W. Baxter and Allen B. Reitz, ReductiveAminations of Carbonyl Compounds with Borohydride and Borane ReducingAgents, Organic Reactions, Chapter 1, pages 1-57 (Wiley, 2002), which isincorporated herein by reference in its entirety. Non-limiting classesof appropriate reducing agents include borohydride, cyanoborohydride,tri(C₁₋₄ acyl)oxyborohydride (e.g., triacetoxyborohydride derivatives),9-borobicyclo[3.3.1]nonane hydride, tri(C₁₋₄ alkyl)borohydride, anddisopinocampteylcyanoborohydride derivatives, amino boranes,borane-pyridine complex, and alkylamine boranes. Non-limiting examplesof appropriate reducing agents include sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium cyano-9-borobicyclo[3.3.1]nonane hydride,tetrabutylammonium cyanoborohydride, cyanoborohydride on a solidsupport, tetramethylammonium triacetoxyborohydride, sodiumtriacetoxyborohydride, lithium triethylborohydride, lithiumtri(sec-butyl)borohydride, sodium disopinocampteylcyanoborohydride,catechol borane, borane tetrahydrofuran, sodium borohydride, potassiumborohydride, lithium borohydride, palladium in the presence of hydrogengas, 5-ethyl-2-methylpyridine borane (PEMB), 2-picoline borane orpolymer-supported triacetoxyborohydride. In some embodiments, any of theaforementioned, and preferably sodium cyanoborohydride, is used incombination with a titanium (IV) additive, dehydrating agent, or a zinchalide additive. In some embodiments, the reducing agent is a tetra(C₁₋₄alkyl)ammonium cyanoborohydride or triacetoxyborohydride, an alkalimetal cyanoborohydride or triacetoxyborohydride, or an alkaline earthcyanoborohydride or triacetoxyborohydride. In some embodiments, thereducing agent is an alkali metal cyanoborohydride. In some embodiments,the reducing agent is selected from sodium cyanoborohydride and sodiumtriacetoxyborohydride. In some embodiments, the reducing agent is sodiumtriacetoxyborohydride. As used herein, a titanium (IV) additive is aLewis acid containing a titanium (IV) metal (e.g., titaniumtetrachloride, titanium isopropoxide, titanium ethoxide, and the like).

In some embodiments, the compound of Formula V, or salt thereof, is2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitiriledihydrochloride salt. In some embodiments, the reacting is carried outin the presence of at least two equivalents of a second base. In someembodiments, the second base is a tertiary amine (e.g., triethylamine).As used herein, “second” in the phrase “second base” is used todifferentiate the base from other bases used in earlier or later stepsof the process and does not indicate that two bases must be present.

In some embodiments, greater than 1 equivalent of the compound ofFormula VI is used based on the compound of Formula V, or salt thereof.

In some embodiments, reaction of a compound of Formula V, or saltthereof, with a compound of Formula VI is performed in dichloromethanesolvent.

In some embodiments, the process further comprises reacting the compoundof Formula I with adipic acid to form the adipate salt of the compoundof Formula I

In another aspect, the present invention provides a compound of FormulaVII:

or a salt thereof; wherein:

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

In some embodiments, the compound of Formula VII is a compound havingFormula VIIa:

or a salt thereof.

The present invention further provides a process for making a compoundof Formula VII, comprising reacting a compound of Formula VIII:

with a compound of Formula IX:

in the presence of a coupling agent to form a compound of Formula VII;wherein:

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

In some embodiments, the process includes a process of making a compoundof Formula VIIa comprise reacting a compound of Formula VIII:

with a compound of Formula IXa:

in the presence of a coupling agent to form a compound of Formula VIIa:

In some embodiments, the coupling agent for the reaction of a compoundof Formula VIII, with a compound of Formula IX or a compound of FormulaIXa, is 1,8-diazabicyclo[5,4,0]undecene. In some embodiments, about 1.05to about 1.2 equivalents (e.g., 1.12 equivalents) of coupling agent isused based on the compound of Formula VIII.

In some embodiments, reacting of the compound of Formula VIII with thecompound of Formula IX or IXa is conducted in a solvent componentcomprising acetonitrile, at a temperature of about 40° C. to about 60°C. In some embodiments, 1 to 1.2 equivalents of the compound of FormulaIX or IXa are used based on the compound of Formula VIII.

In some embodiments, the compound of Formula VIIa is reacted with acompound of Formula IVa:

under Suzuki coupling conditions to form a compound of Formula I:

wherein the Suzuki coupling conditions comprise heating a reactionmixture comprising the compound of Formula VIIa, the compound of FormulaIVa, a Suzuki coupling catalyst, a base and a second solvent component.

In some embodiments, the Suzuki catalyst istetrakis(triphenylphosphine)palladium(0). In some embodiments, the base(e.g., sodium bicarbonate) is present in 4 equivalents or more (e.g., 5equivalents) based on the compound of Formula VII or VIIa.

In some embodiments, the second solvent component comprises 1,4-dioxaneand water, e.g., a 1:1 volume ratio.

In some embodiments, the compounds of Formula VII or VIIa, and IVa, arepresent in about a 1:1 molar ratio.

In some embodiments, the compound of Formula VIIa is reacted with acompound of Formula IVa:

under Suzuki coupling conditions to form a compound of Formula I:

wherein the Suzuki coupling conditions comprise heating a reactionmixture comprising the compound of Formula VIIa, the compound of FormulaIVa, tetrakis(triphenylphosphine)palladium(0), sodium bicarbonate, and asecond solvent component, wherein the second solvent component compriseswater and 1,4-dioxane.

In another aspect, the present invention further provides a compound ofFormula VIII:

or a salt thereof.

In yet another aspect, the present invention provides a process ofpreparing a compound of Formula VIII, or a salt thereof, comprisingreacting a compound of Formula VI:

with a compound of Formula X:

or a salt thereof, in the presence of a reducing agent.

In some embodiments, the compound of Formula X, or salt thereof, is2-(azetidin-3-ylidene)acetonitrile hydrochloride.

In some embodiments, reacting a compound of Formula VI and a compound ofFormula X, or salt thereof, is in the presence of a reducing agent suchas sodium cyanoborohydride or sodium triacetoxyborohydride (e.g., sodiumtriacetoxyborohydride). About 1.5 to about 2.5 equivalents (e.g., 2equivalents) of the reducing agent can be used based on the compound ofFormula X, or salt thereof.

In some embodiments, reacting the compound of Formula VI and thecompound of Formula X, or salt thereof, is conducted in a solventcomponent comprising dichloromethane.

In yet another aspect, the present invention features a compound ofFormula III:

or a salt thereof; wherein:

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

In some embodiments, the compound of Formula III is compound havingFormula IIIa:

or a salt thereof.

In another aspect, the present invention features a process of preparinga compound of Formula III, comprising reacting a compound of Formula X:

or a salt thereof, with a compound of Formula IX:

in the presence of a coupling agent to form a compound of Formula III,or a salt thereof; wherein:

R¹ and R² are each independently H or C₁₋₆ alkyl; or

R¹ and R², together with the two oxygen atoms to which they are attachedand the boron atom to which the oxygen atoms are attached, form a 5- to6-membered heterocycloalkyl ring, which is optionally substituted with1, 2, 3, or 4 C₁₋₄ alkyl groups.

In some embodiments, the coupling agent used in reacting a compound ofFormula X, or salt thereof, with a compound of Formula IX is1,8-diazabicyclo[5,4,0]undecene. In some embodiments, 0.1 to 0.2equivalent of coupling agent is used based on the compound of Formula X,or salt thereof.

In some embodiments, the reacting of the compound of Formula X, or saltthereof, with the compound of Formula IX is conducted in a solventcomponent comprising isopropyl alcohol, for example, at a temperature ofabout 70° C. to about 90° C.

In some embodiments, 1 to 1.1 equivalents of the compound of Formula IXare used based on the compound of Formula X, or salt thereof.

In yet another aspect, the present invention features a process ofpreparing a compound of Formula IIIa, comprising reacting a compound ofFormula X:

with a compound of Formula IXa:

in the presence of a coupling agent to form a compound of Formula III.

In some embodiments, the coupling agent used in reacting a compound ofFormula X with a compound of Formula IXa is1,8-diazabicyclo[5,4,0]undecene. In some embodiments, 0.1 to 0.2equivalent of coupling agent is used based on the compound of Formula X.

In some embodiments, the reacting of the compound of Formula X with thecompound of Formula IXa is conducted in a solvent component comprisingisopropyl alcohol, for example, at a temperature of about 70° C. toabout 90° C.

In some embodiments, 1 to 1.1 equivalents of the compound of Formula IXaare used based on the compound of Formula X.

Uses

The compound of Formula I,{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,is an inhibitor of JAK (e.g., JAK1, JAK2). JAK inhibitors are useful intreating various JAK-associated diseases or disorders. Examples ofJAK-associated diseases include diseases involving the immune systemincluding, for example, organ transplant rejection (e.g., allograftrejection and graft versus host disease). Further examples ofJAK-associated diseases include autoimmune diseases such as multiplesclerosis, rheumatoid arthritis, juvenile arthritis, psoriaticarthritis, type I diabetes, lupus, psoriasis, inflammatory boweldisease, ulcerative colitis, Crohn's disease, myasthenia gravis,immunoglobulin nephropathies, myocarditis, autoimmune thyroid disorders,chronic obstructive pulmonary disease (COPD), and the like. In someembodiments, the autoimmune disease is an autoimmune bullous skindisorder such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

Further examples of JAK-associated diseases include allergic conditionssuch as asthma, food allergies, eszematous dermatitis, contactdermatitis, atopic dermatitis (atropic eczema), and rhinitis. Furtherexamples of JAK-associated diseases include viral diseases such asEpstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1,Varicella-Zoster Virus (VZV) and Human Papilloma Virus (HPV).Further examples of JAK-associated disease include diseases associatedwith cartilage turnover, for example, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, Tietze syndrome, costal athropathy, osteoarthritisdeformans endemica, Mseleni disease, Handigodu disease, degenerationresulting from fibromyalgia, systemic lupus erythematosus, scleroderma,or ankylo sing spondylitis.

Further examples of JAK-associated disease include congenital cartilagemalformations, including hereditary chrondrolysis, chrondrodysplasias,and pseudochrondrodysplasias (e.g., microtia, enotia, and metaphysealchrondrodysplasia). Further examples of JAK-associated diseases orconditions include skin disorders such as psoriasis (for example,psoriasis vulgaris), atopic dermatitis, skin rash, skin irritation, skinsensitization (e.g., contact dermatitis or allergic contact dermatitis).For example, certain substances including some pharmaceuticals whentopically applied can cause skin sensitization. In some embodiments,co-administration or sequential administration of at least one JAKinhibitor of the invention together with the agent causing unwantedsensitization can be helpful in treating such unwanted sensitization ordermatitis. In some embodiments, the skin disorder is treated by topicaladministration of at least one JAK inhibitor of the invention.

Further examples of JAK-associated diseases or conditions include thosecharacterized by solid tumors (e.g., prostate cancer, renal cancer,hepatic cancer, pancreatic cancer, gastric cancer, breast cancer, lungcancer, cancers of the head and neck, thyroid cancer, glioblastoma,Kaposi's sarcoma, Castleman's disease, uterine leiomyosarcoma, melanomaetc.), hematological cancers (e.g., lymphoma, leukemia such as acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) ormultiple myeloma), and skin cancer such as cutaneous T-cell lymphoma(CTCL) and cutaneous B-cell lymphoma. Example CTCLs include Sezarysyndrome and mycosis fungoides. Other examples of JAK-associateddiseases or conditions include pulmonary arterial hypertension.

Other examples of JAK-associated diseases or conditions includeinflammation-associated cancers. In some embodiments, the cancer isassociated with inflammatory bowel disease. In some embodiments, theinflammatory bowel disease is ulcerative colitis. In some embodiments,the inflammatory bowel disease is Crohn's disease. In some embodiments,the inflammation-associated cancer is colitis-associated cancer. In someembodiments, the inflammation-associated cancer is colon cancer orcolorectal cancer. In some embodiments, the cancer is gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),adenocarcinoma, small intestine cancer, or rectal cancer.

JAK-associated diseases can further include those characterized byexpression of: JAK2 mutants such as those having at least one mutationin the pseudo-kinase domain (e.g., JAK2V617F); JAK2 mutants having atleast one mutation outside of the pseudo-kinase domain; JAK1 mutants;JAK3 mutants; erythropoietin receptor (EPOR) mutants; or deregulatedexpression of CRLF2.

JAK-associated diseases can further include myeloproliferative disorders(MPDs) such as polycythemia vera (PV), essential thrombocythemia (ET),myelofibrosis with myeloid metaplasia (MMM), primary myelofibrosis(PMF), chronic myelogenous leukemia (CML), chronic myelomonocyticleukemia (CMML), hypereosinophilic syndrome (HES), systemic mast celldisease (SMCD), and the like. In some embodiments, themyeloproliferative disorder is myelofibrosis (e.g., primarymyelofibrosis (PMF) or post polycythemia vera/essential thrombocythemiamyelofibrosis (Post-PV/Post-ET MF)). In some embodiments, themyeloproliferative disorder is post-essential thrombocythemiamyelofibrosis (Post-ET MF). In some embodiments, the myeloproliferativedisorder is post polycythemia vera myelofibrosis (Post-PV MF). Otherexamples of JAK-associated diseases or conditions include amelioratingthe dermatological side effects of other pharmaceuticals byadministration of the compound of the invention. For example, numerouspharmaceutical agents result in unwanted allergic reactions which canmanifest as acneiform rash or related dermatitis. Example pharmaceuticalagents that have such undesirable side effects include anti-cancer drugssuch as gefitinib, cetuximab, erlotinib, and the like. The compounds ofthe invention can be administered systemically or topically (e.g.,localized to the vicinity of the dermatitis) in combination with (e.g.,simultaneously or sequentially) the pharmaceutical agent having theundesirable dermatological side effect. In some embodiments, thecompound of the invention can be administered topically together withone or more other pharmaceuticals, where the other pharmaceuticals whentopically applied in the absence of a compound of the invention causecontact dermatitis, allergic contact sensitization, or similar skindisorder. Accordingly, compositions of the invention include topicalformulations containing the compound of the invention and a furtherpharmaceutical agent which can cause dermatitis, skin disorders, orrelated side effects.

Further JAK-associated diseases include inflammation and inflammatorydiseases. Example inflammatory diseases include sarcoidosis,inflammatory diseases of the eye (e.g., iritis, uveitis, scleritis,conjunctivitis, or related disease), inflammatory diseases of therespiratory tract (e.g., the upper respiratory tract including the noseand sinuses such as rhinitis or sinusitis or the lower respiratory tractincluding bronchitis, chronic obstructive pulmonary disease, and thelike), inflammatory myopathy such as myocarditis, and other inflammatorydiseases. In some embodiments, the inflammation disease of the eye isblepharitis.

Further JAK-associated diseases include ischemia reperfusion injuries ora disease or condition related to an inflammatory ischemic event such asstroke or cardiac arrest, endotoxin-driven disease state (e.g.,complications after bypass surgery or chronic endotoxin statescontributing to chronic cardiac failure), anorexia, cachexia, fatiguesuch as that resulting from or associated with cancer, restenosis,sclerodermitis, fibrosis, conditions associated with hypoxia orastrogliosis such as, for example, diabetic retinopathy, cancer, orneurodegeneration, and other inflammatory diseases such as systemicinflammatory response syndrome (SIRS) and septic shock.

Other JAK-associated diseases include gout and increased prostate sizedue to, e.g., benign prostatic hypertrophy or benign prostatichyperplasia, as well as bone resorption diseases such as osteoporosis orosteoarthritis, bone resorption diseases associated with: hormonalimbalance and/or hormonal therapy, autoimmune disease (e.g. osseoussarcoidosis), or cancer (e.g. myeloma).

Further JAK-associated diseases include a dry eye disorder. As usedherein, “dry eye disorder” is intended to encompass the disease statessummarized in a recent official report of the Dry Eye Workshop (DEWS),which defined dry eye as “a multifactorial disease of the tears andocular surface that results in symptoms of discomfort, visualdisturbance, and tear film instability with potential damage to theocular surface. It is accompanied by increased osmolarity of the tearfilm and inflammation of the ocular surface.” Lemp, “The Definition andClassification of Dry Eye Disease: Report of the Definition andClassification Subcommittee of the International Dry Eye Workshop”, TheOcular Surface, 5(2), 75-92 April 2007, which is incorporated herein byreference in its entirety. In some embodiments, the dry eye disorder isselected from aqueous tear-deficient dry eye (ADDE) or evaporative dryeye disorder, or appropriate combinations thereof. In some embodiments,the dry eye disorder is Sjogren syndrome dry eye (SSDE). In someembodiments, the dry eye disorder is non-Sjogren syndrome dry eye(NSSDE).

Further JAK-associated diseases include conjunctivitis, uveitis(including chronic uveitis), chorioditis, retinitis, cyclitis,sclieritis, episcleritis, or iritis. Other JAK-associated diseasesinclude respiratory dysfunction or failure associated wth viralinfection, such as influenza and SARS.

EXAMPLES

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

Example 1. Synthesis of2-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrileAdipate (9)

tert-Butyl3-(cyanomethyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate(3)

To a 1-L flask equipped with a nitrogen inlet, a thermocouple, and amechanical stirrer were sequentially added isopropanol (IPA, 200 mL),1,8-diazabicyclo[5,4,0]undec-ene (DBU, 9.8 g, 64.4 mmol, 0.125 equiv),4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1, 101 g,520.51 mmol, 1.01 equiv) and tert-butyl3-(cyanomethylene)azetidine-1-carboxylate (2, 100 g, 514.85 mmol) atambient temperature to generate a reaction mixture as a suspension. Theresulting reaction mixture was heated to reflux in 30 minutes to providea homogenous solution and the mixture was maintained at reflux for anadditional 2-3 hours. After the reaction was complete as monitored byHPLC, n-heptane (400 mL) was gradually added to the reaction mixture in45 minutes while maintaining the mixture at reflux. Solids wereprecipitated out during the n-heptane addition. Once n-heptane additionwas complete, the mixture was gradually cooled to ambient temperatureand stirred at ambient temperature for an additional 1 hour. The solidswere collected by filtration, washed with n-heptane (200 mL), and driedunder vacuum at 50° C. with nitrogen sweeping to constant weight toafford tert-butyl 3-(cyanomethyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate(3, 181 g, 199.9 g theoretical, 90.5%) as a white to pale yellow solid.For 3: ¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (s, 1H), 7.74 (s, 1H), 4.45-4.23(m, 2H), 4.23-4.03 (m, 2H), 3.56 (s, 2H), 1.38 (s, 9H), 1.25 (s, 12H)ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ 155.34, 145.50, 135.88, 116.88, 107.08(br), 83.15, 79.36, 58.74 (br), 56.28, 27.96, 26.59, 24.63 ppm;C₁₉H₂₉BN₄O₄ (MW 388.27), LCMS (EI) m/e 389 (M⁺+H).

tert-Butyl3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)-azetidine-1-carboxylate(5)

To a 1-L flask equipped with a nitrogen inlet, a thermocouple, and amechanical stirrer were added 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (4,39.6 g, 257.6 mmol), tert-butyl3-(cyanomethyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate(3, 100 g, 257.6 mmol, 1.0 equiv), cesium fluoride (136.9 g, 901.4 mmol,3.5 equiv), tert-butanol (250 mL), water (250 mL), and[1,1′-bis(di-cyclohexylphosphino)ferrocene]dichloropalladium(II)(Pd-127, 351.4 mg, 0.46 mmol, 0.0018 equiv) at ambient temperature. Theresulting reaction mixture was de-gassed and refilled with nitrogen for3 times before being heated to reflux and maintained at reflux undernitrogen for 20-24 hours. When HPLC showed the reaction was complete,the reaction mixture was cooled to 45-55° C. in 30 minutes, the twophases were separated, and the aqueous phase was discarded. To theorganic phase was added n-heptane (125 mL) in 30 minutes at 45-55° C.The resulting mixture was slowly cooled to ambient temperature in onehour and stirred at ambient temperature for an additional 2 hours. Thesolids were collected by filtration, washed with n-heptane (100 mL), anddried under vacuum at 50° C. with nitrogen sweeping to constant weightto afford tert-butyl3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)-azetidine-1-carboxylate(5, 96.8 g, 97.7 g theoretical, 99%) as a pale yellow solid. For 5: ¹HNMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.68 (s, 1H), 8.44 (s, 1H), 7.60(d, J=3.5 Hz, 1H), 7.06 (d, J=3.6 Hz, 1H), 4.62-4.41 (m, 2H), 4.31-4.12(m, 2H), 3.67 (s, 2H), 1.39 (s, 9H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ155.40, 152.60, 150.63, 149.15, 139.76, 129.53, 127.65, 122.25, 116.92,113.21, 99.71, 79.45, 58.34 (br), 56.80, 27.99, 26.83 ppm; C₁₉H₂₁N₇O₂(MW 379.4), LCMS (EI) m/e 380 (M⁺+H).

2-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitriledihydrochloride salt (6)

To a 0.5-L flask equipped with a nitrogen inlet, a thermocouple, anadditional funnel, and a mechanical stirrer were added tert-butyl3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azetidine-1-carboxylate(5, 15 g, 39.5 mmol), water (7.5 mL, 416 mmol) and dichloromethane (75mL) at room temperature. The mixture was stirred at room temperature togenerate a suspension. To the suspension was added a solution of 5 Mhydrogen chloride (HCl) in isopropanol (55 mL, 275 mmol, 7.0 equiv) in 5minutes. The resulting reaction mixture was then heated to gentle refluxand maintained at reflux for 3-4 hours. After the reaction was completedas monitored by HPLC, tert-butyl methyl ether (TBME, 45 mL) was added tothe reaction suspension. The mixture was gradually cooled to roomtemperature, and stirred for an additional one hour. The solids werecollected by filtration, washed with tert-butyl methyl ether (TBME, 45mL) and dried under vacuum at 50° C. with nitrogen sweeping to constantweight to afford2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitriledihydrochloride salt (6, 13.6 g, 13.9 g theoretical, 98%) as anoff-white to light yellow solid. For 6: ¹H NMR (400 MHz, D₂O) δ 8.96 (s,1H), 8.81 (s, 1H), 8.49 (s, 1H), 7.78 (d, J=3.8 Hz, 1H), 7.09 (d, J=3.7Hz, 1H), 4.93 (d, J=12.8 Hz, 2H), 4.74 (d, J=12.5 Hz, 2H), 3.74 (s, 2H)ppm; ¹³C NMR (101 MHz, D₂O) δ 151.35, 143.75, 143.33, 141.33, 132.03,131.97, 115.90, 114.54, 113.85, 103.18, 59.72, 54.45 (2C), 27.02 ppm;C₁₄H₁₅Cl₂N₇ (C₁₄H₁₃N₇ for free base, MW 279.30), LCMS (EI) m/e 280(M⁺+H).

2-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile(8, Free Base)

To a 0.5-L flask equipped with a nitrogen inlet, a thermocouple, anadditional funnel, and a mechanical stirrer were added2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitriledihydrochloride salt (6, 20 g, 56.78 mmol), dichloromethane (200 mL) andtriethylamine (TEA, 16.62 mL, 119.2 mmol, 2.1 equiv) at ambienttemperature. The mixture was stired at ambient temperature for 30minutes before1-(3-fluoro-2-(trifluoromethyl)-isonicotinoyl)piperidin-4-one (7, 17.15g, 57.91 mmol, 1.02 equiv) was added to the mixture. The mixture wasthen treated with sodium triacetoxyborohydride (25.34 g, 113.6 mmol, 2.0equiv) in 5 minutes at ambient temperature (below 26° C.). The resultingreaction mixture was stirred at ambient temperature for 2 hours. Afterthe reaction was complete as monitored by HPLC, the reaction mixture wasquenched with saturated NaHCO₃ aqueous solution (200 mL). The two phaseswere separated and the aqueous phase was extracted with methylenechloride (200 mL). The combined organic phase was washed with 4% brine(100 mL) followed by solvent switch of methylene chloride to acetone bydistillation. The resulting solution of the desired crude product (8) inacetone was directly used for the subsequent adipate salt formation. Asmall portion of solution was purified by column chromatography (SiO₂,0-10% of MeOH in EtOAc gradient elution) to afford the analytically pure2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile(8 free base) as an off-white solid. For 8: ¹H NMR (400 MHz, DMSO-d₆) δ12.17 (d, J=2.8 Hz, 1H), 8.85 (s, 1H), 8.70 (m, 2H), 8.45 (s, 1H), 7.93(t, J=4.7 Hz, 1H), 7.63 (dd, J=3.6, 2.3 Hz, 1H), 7.09 (dd, J=3.6, 1.7Hz, 1H), 4.10 (m, 1H), 3.78 (d, J=7.9 Hz, 2H), 3.61 (t, J=7.9 Hz, 1H),3.58 (s, 2H), 3.46 (m, 1H), 3.28 (t, J=10.5 Hz, 1H), 3.09 (ddd, J=13.2,9.5, 3.1 Hz, 1H), 2.58 (m, 1H), 1.83-1.75 (m, 1H), 1.70-1.63 (m, 1H),1.35-1.21 (m, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ 160.28, (153.51,150.86), 152.20, 150.94, 149.62, (146.30, 146.25), 139.48, (134.78,134.61), (135.04, 134.92, 134.72, 134.60, 134.38, 134.26, 134.03,133.92), 129.22, 127.62, 126.84, 121.99, 122.04, (124.77, 122.02,119.19, 116.52), 117.39, 113.00, 99.99, 61.47, 60.49, 57.05, 44.23,28.62, 27.88, 27.19 ppm; C₂₆H₂₃F₄N₉O (MW, 553.51), LCMS (EI) m/e 554.1(M⁺+H).

2-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrileAdipate (9)

To a 0.5-L flask equipped with a mechanical stirrer, a thermocouple, anaddition funnel, and a nitrogen inlet was added a solution of crude2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile(8 free base, 31.38 g, 56.7 mmol) in acetone (220 mL) and adipic acid(8.7 g, 59.53 mmol, 1.05 equiv) at ambient temperature. The reactionmixture was then heated to reflux to give a solution. n-Heptane (220 mL)was gradually added to the reaction mixture at 40-50° C. in one hour.The resulting mixture was gradually cooled to ambient temperature in onehour and stirred at ambient temperature for an additional 16 hours. Thesolids were collected by filtration, washed with n-heptane (2×60 mL),and dried under vacuum at 50° C. with nitrogen sweeping to constantweight to afford2-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrileadipate (9, 34.0 g, 39.7 g theoretical, 85.6% for two steps) as a whiteto off-white solid. 9: ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H), 12.05(brs, 2H), 8.85 (s, 1H), 8.72 (s, 1H), 8.69 (d, J=4.7 Hz, 1H), 8.45 (s,1H), 7.93 (t, J=4.7 Hz, 1H), 7.63 (dd, J=3.6, 2.3 Hz, 1H), 7.09 (dd,J=3.6, 1.7 Hz, 1H), δ 4.11 (dt, J=11.0, 4.4 Hz, 1H), 3.77 (d, J=7.8 Hz,2H), 3.60 (t, J=7.8 Hz, 2H), 3.58 (s, 2H), 3.44 (dt, J=14.4, 4.6 Hz,1H), 3.28 (t, J=10.4 Hz, 1H), 3.09 (ddd, J=13.2, 9.6, 3.2 Hz, 1H), 2.58(tt, J=8.6, 3.5 Hz, 1H), 2.28-2.17 (m, 4H), 1.83-1.74 (m, 1H), 1.67 (d,J=11.0 Hz, 1H), 1.59-1.46 (m, 4H), 1.37-1.21 (m, 2H) ppm; ¹³C NMR (101MHz, DMSO-d₆) δ 174.38, 160.29, (153.52, 150.87), 152.20, 150.94,149.63, (146.30, 146.25), 139.48, (134.79, 134.62), (135.08, 134.97,134.74, 134.62, 134.38, 134.28, 134.04, 133.93), 129.21, 127.62, 126.84,122.05, (124.75, 122.02, 119.29, 116.54), 117.39, 113.01, 99.99, 61.47,60.50, 57.06, 44.24, 33.42, 30.70, 28.63, 27.89, 27.20, 24.07 ppm;C₃₂H₃₃F₄N₉O₅ (MW 699.66; C₂₆H₂₃F₄N₉O for free base, MW, 553.51), LCMS(EI) m/e 554.0 (M⁺+H).

Example 2: Alternative Synthesis of2-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

2-(Azetidin-3-ylidene)acetonitrile hydrochloride (2a)

To a 0.5-L flask equipped with a nitrogen inlet, a thermocouple, and amechanical stirrer were added tert-butyl3-(cyanomethylene)azetidine-1-carboxylate (2, 30 g, 154.46 mmol) andmethylenechloride (300 mL) at ambient temperature. The solution was thentreated with a solution of 5 M hydrogen chloride (HCl) in isopropanolsolution (294.2 mL, 1.54 mol, 10 equiv) at ambient temperature and theresulting reaction mixture was stirred at ambient temperature for 18hours. After the reaction was complete as monitored by HPLC, thesuspension was added tert-butyl methyl ether (TBME, 150 mL), and themixture was stirred at ambient temperature for 2 hours. The solids wascollected by filtration, washed with n-heptane (2×100 mL), and dried onthe filtration funnel at ambient temperature for 3 hours to afford2-(azetidin-3-ylidene)acetonitrile hydrochloride (2a, 13.7 g, 20.2 gtheoretical, 67.8%) as a white solid. For 2a: ¹H NMR (500 MHz, DMSO-d₆)δ 9.99 (s, 2H), 5.94 (p, J=2.5 Hz, 1H), 4.85-4.80 (m, 2H), 4.77-4.71 (m,2H) ppm; ¹³C NMR (126 MHz, DMSO-d₆) δ 155.65, 114.54, 94.78, 55.26,54.63 ppm; C₅H₇ClN₂ (MW 130.58; C₅H₆N₂ for free base, MW 94.11), LCMS(EI) m/e 95 (M⁺+H).

2-(1-(1-(3-Fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-ylidene)acetonitrile(10)

To a 0.25-L flask equipped with a nitrogen inlet, a thermocouple, and amagnetic stirrer were added 2-(azetidin-3-ylidene)acetonitrilehydrochloride (2a, 4.5 g, 34.46 mmol),1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-one (7, 10 g,34.46 mmol, 1.0 equiv), and methylenechloride (100 mL) at ambienttemperature and the resulting mixture was then treated with sodiumtriacetoxyborohydride (14.6 g, 68.93 mmol, 2.0 equiv) at ambienttemperature. The reaction mixture was stirred at ambient temperature for2 hours before being quenched with saturated sodium bicarbonate (NaHCO₃)aqueous solution (50 mL). The two phases were separated and the aqueousphase was extracted with dichloromethane (200 mL). The combined organicphase was washed with water (50 mL) and brine (50 mL) and concentratedunder reduced pressure to afford the crude desired product (10), whichwas purified by column chromatography (SiO₂, 0-10% of ethyl acetate inhexane gradient elution) to afford2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-ylidene)acetonitrile(10, 9.5 g, 12.7 g theoretical, 74.8%) as a white solid. For 10: ¹H NMR(400 MHz, CDCl₃) δ 8.57 (d, J=4.7 Hz, 1H), 7.54 (t, J=4.6 Hz, 1H), 5.29(p, J=2.4 Hz, 1H), 4.18-4.08 (m, 1H), 4.08-4.03 (m, 2H), 3.98-3.94 (m,2H), 3.57-3.39 (m, 2H), 3.17-3.04 (m, 1H), 2.56 (tt, J=7.4, 3.5 Hz, 1H),1.86-1.77 (m, 1H), 1.75-1.64 (m, 1H), 1.54-1.43 (m, 1H), 1.43-1.31 (m,1H) ppm; ¹³C NMR (101 MHz, CDCl₃) δ 161.34, 160.73, 152.62 (d, J=269.1Hz), 145.75 (d, J=6.1 Hz), 136.73 (qd, J=36.1, 12.0 Hz), 134.56 (d,J=16.9 Hz), 126.89, 120.58 (qd, J=275.0, 4.9 Hz), 115.11, 92.04, 62.05,60.57 (2C), 44.47, 39.42, 29.38, 28.47 ppm; C₁₇H₁₆F₄N₄O (MW 368.33),LCMS (EI) m/e 369 (M⁺+H).

2-(1-(1-(3-Fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitrile(11)

To a 25 mL flask equipped with a nitrogen inlet, a thermocouple, and amagnetic stirrer were added4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1, 210 mg,1.08 mmol, 1.08 equiv),2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-ylidene)acetonitrile(10, 370 mg, 1.0 mmol) and acetonitrile (3 mL) at ambient temperature.The solution was then treated with 1,8-diazabicyclo[5,4,0]undecene (DBU,173 mg, 0.17 mL, 1.12 mmol, 1.12 equiv) at ambient temperature and theresulting reaction mixture was warmed to 50° C. and stirred at 50° C.for overnight. When the reaction was complete as monitored by HPLC, thereaction mixture was directly load on a solica gel (SiO₂) column forchromatographic purification (0-2.5% MeOH in ethyl acetate gradientelution) to afford2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitrile(11, 263 mg, 562.4 mg theoretical, 46.7%) as a white solid. For 11: ¹HNMR (400 MHz, DMSO-d₆) δ 8.64 (d, J=4.7 Hz, 1H), 8.22 (d, J=0.6 Hz, 1H),7.88 (dd, J=4.7 Hz, 1H), 7.69 (s, 1H), 4.10-3.99 (m, 1H), 3.58 (d, J=7.8Hz, 2H), 3.52-3.42 (m, 2H), 3.44 (s, 2H), 3.41-3.33 (m, 1H), 3.28-3.15(m, 1H), 3.03 (ddd, J=12.9, 9.2, 3.2 Hz, 1H), 2.51-2.44 (m, 1H),1.77-1.66 (m, 1H), 1.64-1.54 (m, 1H), 1.28-1.17 (m, 2H), 1.24 (s, 12H)ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ 160.22, 152.13 (d, J=265.8 Hz), 146.23(d, J=5.7 Hz), 145.12, 135.41, 134.66 (d, J=16.9 Hz), 134.43 (qd,J=35.0, 11.7 Hz), 127.58, 120.61 (qd, J=274.4, 4.6 Hz), 117.35, 106.59(br), 83.10, 61.40, 60.53 (2C), 56.49, 44.17, 38.99, 28.55, 27.82,27.02, 24.63 ppm; C₂₆H₃₁BF₄N₆O₃ (MW 562.37), LCMS (EI) m/e 563 (M⁺+H).

2-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile(8)

To a 25-mL flask equipped with a nitrogen inlet, a thermocouple, anadditional funnel, and a magnetic stirrer were added2-(1-(1-(3-fluoro-2-(trifluoromethyl)-isonicotinoyl)piperidin-4-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitrile(11, 307 mg, 0.546 mmol), 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (4, 84.8mg, 0.548 mmol, 1.0 equiv), sodium bicarbonate (NaHCO₃, 229 mg, 2.72mmol, 5.0 equiv), water (1.6 mL), and 1,4-dioxane (1.6 mL) at ambienttemperature. The mixture was then teated withtetrakis(triphenylphosphine)palladium(0) (12.8 mg, 0.011 mmol, 0.02equiv) at ambient temperature and the resulting reaction mixture wasde-gassed and refilled with nitrogen for 3 times before being heated to85° C. The reaction mixture was stired at 85° C. under nitrogen forovernight. When the reaction was complete as monitored by HPLC, thereaction mixture was concentrated to dryness under reduced pressure andthe desired product,2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile(8 free base, 135 mg, 302.2 mg theoretical, 44.6%), was obtained asoff-white solids by direct silica gel (SiO₂) column chromatography(0-10% of ethyl acetate in hexane gradient elution) purification of thedried reaction mixture. The compound obtained by this synthetic approachis identical in every comparable aspect to the compound 8 manufacturedby the synthetic method as described above in Example 1.

Example 3. Synthesis of (3-Fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone

(3-Fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone(14)

To a 30 L reactor equipped with a mechanic stirrer, an addition funneland a septum was charged sodium hydroxide (NaOH, 1.4 kg, 35 mol, 2.0equiv) and water (7 L) and the resulting solution was treated with1,4-dioxa-8-azaspiro[4.5]decane hydrochloride (3.13 kg, 17.43 mol) atambient temperature. The resulting mixture was then stirred at ambienttemperature for 30 minutes before being saturated with solid sodiumchloride (1.3 kg) and extracted with 2-methyl-tetrahydrofuran (3×7 L).The combined organic phase was dried with anhydrous sodium sulfate(Na₂SO₄, 1.3 kg) and concentrated under reduced pressure (70 mmHg) at50° C. after removal of the drying reagent, sodium sulfate (Na₂SO₄), byfiltration. The yellow oil thus obtained was distilled under reducedpressure (80 mmHg, by 115 to 120° C.) to afford1,4-dioxa-8-azaspiro[4.5]decane (2.34 kg, 2.496 kg theoretical, 93.8%)as a clear oil, which was used directly in the subsequent couplingreaction.

To a dried 100 L reactor equipped with a mechanic stirrer, an additionfunnel, a thermometer and a vacuum outlet was charged3-fluoro-2-(trifluoromethyl)isonicotinic acid (13, 3.0 kg, 14.35 mol),benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent, 7.6 kg, 17.2 mol, 1.2 equiv),1,4-dioxa-8-azaspiro[4.5]decane (2.34 kg, 16.36 mol, 1.14 equiv) andN,N-dimethylformamide (DMF, 18 L) at ambient temperature. The resultingsolution was then stirred at ambient temperature for 20 minutes beforebeing cooled to 5 to 10° C. Triethylamine (Et₃N, 4 L, 28.67 mol, 2.0equiv) was then added to the reaction mixture over 1 hour and theinternal temperature was kept between 5° C. and 10° C. during theaddition of triethylamine. The dark brown solution thus obtained wasstirred for 12 h at ambient temperature (approximately 20° C.) and thenchilled to around 10° C. With vigorous stirring, 18 L of the saturatedsodium bicarbonate (NaHCO₃) aqueous solution and 36 L of water weresequentially added to the chilled reaction mixture and the internaltemperature was kept under 15° C. The precipitation (filter cake) thusobtained was collected by filtration. The aqueous phase was thensaturated with 12 kg of solid sodium chloride (NaCl) and extracted withEtOAc (2×18 L). The combined organic layer was washed with saturatedsodium bicarbonate (NaHCO₃) aqueous solution (18 L), and water (2×18 L)in sequence. The filter cake collected was then dissolved back in theorganic phase and the resulting dark brown solution was washed withwater (2×18 L) before being concentrated under reduced pressure (40-50°C., 30 mm Hg) to afford approximately 5.0 kg of the crude desiredproduct (14) as a viscous brown oil. The crude product obtained abovewas then dissolved in EtOH (8.15 L) at 50° C. and the resulting solutionwas treated with water (16.3 L) over 30 minutes at around 50° C. Thebrown solution was seeded before being gradually cooled to ambienttemperature (approximately 20° C.) over 3 hours with stirring andstirred at ambient temperature for 12 h. The solids were collected byfiltration, washed with a mixture of EtOH and water (EtOH:H₂O=1:20, 2 L)and dried under reduced pressure (50 mmHg) at approximately 60° C. for24 h to afford(3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone(14, 3.98 kg, 4.797 kg theoretical, 83.0%) as a white solid. For 14: ¹HNMR (300 MHz, DMSO-d₆) δ 8.64 (d, ³J_(HH)=4.68 Hz, 1H, NCH in pyridine),7.92 (dd, ³J_(HH)=4.68 Hz, ⁴J_(HF)=4.68 Hz, 1H, NCCH in pyridine),3.87-3.91 (m, 4H, OCH₂CH₂O), 3.70 (br s, 2H, one of NCH₂ in piperidinering, one of another NCH₂ in piperidine ring, both in axial position),3.26 (t, ³J_(HH)=5.86 Hz, 2H, one of NCH₂ in piperidine ring, one ofanother NCH₂ in piperidine ring, both in equatorial position), 1.67 (d,³J_(HH)=5.86 Hz, 2H, one of NCCH₂ in piperidine ring, one of anotherNCCH₂ in piperidine ring, both in equatorial position), 1.58 (br s, 2H,one of NCCH₂ in piperidine ring, one of another NCCH₂ in piperidinering, both in axial position) ppm; ¹³C NMR (75 MHz, DMSO-d₆) δ 161.03(N—C═O), 151.16 (d, ¹J_(CF)=266.03 Hz, C—F), 146.85 (d, ⁴J_(CF)=4.32 Hz,NCH in pyridine), 135.24 (d, ²J_(CF)=11.51 Hz, C—C═O), 135.02 (quartet,²J_(CF)=34.57 Hz, NCCF₃), 128.24 (d, ⁴J_(CF)=7.48 Hz, NCCH in pyridine),119.43 (d×quartet, ¹J_(CF)=274.38 Hz, ³J_(CF)=4.89 Hz, CF₃), 106.74(OCO), 64.60 (OCCO), 45.34 (NC in piperidine ring), 39.62 (NC inpiperidine ring), 34.79 (NCC in piperidine ring), 34.10 (NCC inpiperidine ring) ppm; ¹⁹F NMR (282 MHz, DMSO-d₆) δ −64.69 (d,⁴J_(FF)=15.85 Hz, F₃C), −129.26 (d×quartet, ⁴J_(FF)=15.85 Hz,⁴J_(FH)=3.96 Hz, FC) ppm; C₁₄H₄F₄N₂O₃ (MW, 334.27), LCMS (EI) m/e 335.1(M⁺+H).

(3-Fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone (7)

In a 5 L 4-necked round bottom flask equipped with a mechanical stirrer,a thermocouple, an addition funnel and a nitrogen inlet was charged(3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone(14, 100 g, 0.299 mol) in acetonitrile (ACN, 400 mL) at ambienttemperature. The resultant solution was cooled to below 10° C. beforebeing treated with 6.0 N aqueous hydrochloric acid (HCl) solution (450mL, 2.70 mol, 9.0 equiv) while the internal temperature was kept atbelow 10° C. The resulting reaction mixture was then gradually warmed toroom temperature and an additional amount of 6.0 N aqueous hydrochloricacid (HCl) solution (1050 mL, 6.30 mol, 21.0 equiv) was slowlyintroduced to the reaction mixture at ambient temperature over 8 hoursvia the addition funnel. When the reaction was complete as monitored byHPLC, the reaction mixture was then cooled to 0° C. before being treatedwith 30% aqueous sodium hydroxide (NaOH, 860 mL, 8.57 mmol, 28.6 equiv)while the internal temperature was kept at below 10° C. The resultingreaction mixture was subsequently warmed to ambient temperature prior toaddition of solid sodium bicarbonate (NaHCO₃, 85.0 g, 1.01 mol, 3.37equiv) over 1 hour. The mixture was then extracted with EtOAc (2×1.2 L),and the combined organic phase was washed with 16% aqueous sodiumchloride solution (2×800 mL) and concentrated to approximately 1.0 L byvacuum distillation. n-Heptane (2.1 L) was added to the residue, and theresulting mixture was concentrated to 1.0 L by vacuum distillation. Tothe concentrated mixture was added n-heptane (2.1 L). The resultingwhite slurry was then concentrated to 1.0 L by vacuum distillation. Tothe white slurry was then added methyl tert-butyl ether (MTBE, 1.94 L).The white turbid was heated to 40° C. to obtain a clear solution. Theresulting solution was concentrated to about 1.0 L by vacuumdistillation. The mixture was stirred at room temperature for 1 hour.The white precipitate was collected by filtration, washed with n-heptane(400 mL) and dried on the filter under nitrogen with pulling vacuum toafford (3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone (7, 78.3 g, 86.8 gtheoretical, 90.2%) as an off-white solid. For 7: ¹H NMR (300 MHz,DMSO-d₆) δ 8.68 (d, ³J_(HH)=4.69 Hz, 1H, NCH in pyridine), 7.97 (dd,³J_(HH)=4.69 Hz, ⁴J_(HF)=4.69 Hz, 1H, NCCH in pyridine), 3.92 (br s, 2H,one of NCH₂ in piperidine ring, one of another NCH₂ in piperidine ring,both in axial position), 3.54 (t, ³J_(HH)=6.15 Hz, 2H, one of NCH₂ inpiperidine ring, one of another NCH₂ in piperidine ring, both inequatorial position), 2.48 (t, ³J_(HH)=6.44 Hz, 2H, NCCH₂), 2.34 (t,³J_(HH)=6.15 Hz, 2H, NCCH₂) ppm; ¹³C NMR (75 MHz, DMSO-d₆) δ 207.17(C═O), 161.66 (N—C═O), 151.26 (d, ¹J_(CF)=266.89 Hz, C—F), 146.90 (d,³J_(CF)=6.05 Hz, NCH in pyridine), 135.56 (C—C═O), 134.78-135.56 (m,NCCF₃), 128.27 (d, ³J_(CF)=7.19 Hz, NCCH in pyridine), 119.52(d×quartet, ¹J_(CF)=274.38 Hz, ³J_(CF)=4.89 Hz, CF₃), 45.10 (NC inpiperidine ring) ppm, one carbon (NCC in piperidine ring) missing due tooverlap with (CD₃)₂SO; ¹⁹F NMR (282 MHz, DMSO-d₆) δ −64.58 (d,⁴J_(FF)=15.85 Hz, F₃C), −128.90 (d×quartet, ⁴J_(FF)=1 5.85 Hz,⁴J_(FH)=4.05 Hz, FC) ppm; C₁₂H₁₀F₄N₂O₂ (MW, 290.21), LCMS (EI) m/e 291.1(M⁺+H).

Example 4. Synthesis of tert-Butyl3-(cyanomethylene)azetidine-1-carboxylate Example IV

1-Benzhydrylazetidin-3-ol hydrochloride (16)

A solution of diphenylmethanamine (2737 g, 15.0 mol, 1.04 equiv) inmethanol (MeOH, 6 L) was treated with 2-(chloromethyl)oxirane (1330 g,14.5 mol) from an addition funnel at ambient temperature. During theinitial addition a slight endotherm was noticed. The resulting reactionmixture was stirred at room temperature for 3 days before being warmedto reflux for an additional 3 days. When TLC showed that the reactionwas deemed complete, the reaction mixture was first cooled down to roomtemperature and then to 0-5° C. in an ice bath. The solids werecollected by filtration and washed with acetone (4 L) to give the firstcrop of the crude desired product (1516 g). The filtrate wasconcentrated under reduced pressure and the resulting semisolid wasdiluted with acetone (1 L). This solid was then collected by filtrationto give the second crop of the crude desired product (221 g). The crudeproduct, 1-benzhydrylazetidin-3-ol hydrochloride (1737 g, 3998.7 gtheoretical, 43.4% yield), was found to be sufficiently pure to be usedin the subsequent reaction without further purification. ¹HNMR (300 MHz,DMSO-d₆) δ 12.28 (br. d, 1H), 7.7 (m, 5H), 7.49 (m, 5H), 6.38 (d, 1H),4.72 (br. s, 1H), 4.46 (m, 1H), 4.12 (m, 2H), 3.85 (m, 2H) ppm;C₁₆H₁₈ClNO (MW 275.77; C₁₆H₁₇NO for free base, MW, 239.31), LCMS (EI)m/e 240 (M⁺+H).

tert-Butyl 3-hydroxyazetidine-1-carboxylate (17)

A suspension of 1-benzhydrylazetidin-3-ol hydrochloride (625 g, 2.27mol) in a 10% solution of aqueous sodium carbonate (Na₂CO₃, 5 L) anddichloromethane (CH₂Cl₂, 5 L) was stirred at room temperature until allsolids were dissolved. The two layers were separated, and the aqueouslayer was extracted with dichloromethane (CH₂Cl₂, 2 L). The combinedorganics extracts were dried over sodium sulfate (Na₂SO₄) andconcentrated under reduced pressure. The resulting crude1-benzhydrylazetidin-3-ol free base was then dissolved in THF (6 L) andthe solution was placed into a large Parr bomb. Di-tert-butyldicarbonate (BOC₂O, 545 g, 2.5 mol, 1.1 equiv) and 20% palladium (Pd) oncarbon (125 g, 50% wet) were added to the Parr bomb. The vessel wascharged to 30 psi with hydrogen gas (H₂) and stirred under steadyhydrogen atmosphere (vessel was recharged three times to maintain thepressure at 30 psi) at room temperature for 18 h. When HPLC showed thatthe reaction was complete (no more hydrogen was taken up), the reactionmixture was filtered through a Celite pad and the Celite pad was washedwith THF (4 L). The filtrates were concentrated under reduced pressureto remove the solvent and the residue was loaded onto a Biotage 150column with a minimum amount of dichloromethane (CH₂Cl₂). The column waseluted with 20-50% ethyl acetate in n-heptane and the fractionscontaining the pure desired product, tert-butyl3-hydroxyazetidine-1-carboxylate, were collected and combined. Thesolvents were removed under reduced pressure to afford tert-butyl3-hydroxyazetidine-1-carboxylate (357 g, 393.2 g theoretical, 90.8%yield) as a colorless oil, which solidified upon standing at ambienttemperature in vacuum. ¹HNMR (300 MHz, CDCl₃), δ 4.56 (m 1H), 4.13 (m,2H), 3.81 (m, 2H), 1.43 (s, 9H) ppm.

tert-Butyl 3-oxoazetidine-1-carboxylate (18)

A solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (50 g, 289mmol) in ethyl acetate (400 mL) was cooled to 0° C. The resultingsolution was then treated with solid TEMPO (0.5 g, 3.2 mmol, 0.011equiv) and a solution of potassium bromide (KBr, 3.9 g, 33.2 mmol, 0.115equiv) in water (60 mL) at 0-5° C. While keeping the reactiontemperature between 0-5° C., a solution of saturated aqueous sodiumbicarbonate (NaHCO₃, 450 mL) and an aqueous sodium hypochlorite solution(NaClO, 10-13% available chlorine, 450 mL) were added. Once the solutionof sodium hypochlorite was added, the color of the reaction mixture waschanged immediately. When additional amount of sodium hypochloritesolution was added, the color of the reaction mixture was graduallyfaded. When TLC showed that all of the starting material was consumed,the color of the reaction mixture was no longer changed. The reactionmixture was then diluted with ethyl acetate (EtOAc, 500 mL) and twolayers were separated. The organic layer was washed with water (500 mL)and the saturated aqueous sodium chloride solution (500 mL) and driedover sodium sulfate (Na₂SO₄). The solvent was then removed under reducedpressure to give the crude product, tert-butyl3-oxoazetidine-1-carboxylate (48 g, 49.47 g theoretical, 97% yield),which was found to be sufficiently pure and was used directly in thesubsequent reaction without further purification. ¹HNMR (CDCl₃, 300 MHz)δ 4.65 (s, 4H), 1.42 (s, 9H) ppm.

tert-Butyl 3-(cyanomethylene)azetidine-1-carboxylate (2)

Diethyl cyanomethyl phosphate (745 g, 4.20 mol, 1.20 equiv) andanhydrous tetrahydrofuran (THF, 9 L) were added to a four-neck flaskequipped with a thermowell, an addition funnel and the nitrogenprotection tube at room temperature. The solution was cooled with anice-methanol bath to −14° C. and a 1.0 M solution of potassiumtert-butoxide (t-BuOK) in anhydrous tetrahydrofuran (THF, 3.85 L, 3.85mol, 1.1 equiv) was added over 20 min keeping the reaction temperaturebelow −5° C. The resulting reaction mixture was stirred for 3 h at −10°C. and a solution of 1-tert-butoxycarbonyl-3-azetidinone (600 g, 3.50mol) in anhydrous tetrahydrofuran (THF, 2 L) was added over 2 h keepingthe internal temperature below −5° C. The reaction mixture was stirredat −5 to −10° C. over 1 h and then slowly warmed up to room temperatureand stirred at room temperature for overnight. The reaction mixture wasthen diluted with water (4.5 L) and saturated aqueous sodium chloridesolution (NaCl, 4.5 L) and extracted with ethyl acetate (EtOAc, 2×9 L).The combined organic layers were washed with brine (6 L) and dried overanhydrous sodium sulfate (Na₂SO₄). The solvent was removed under reducedpressure and the residue was diluted with dichloromethane (CH₂Cl₂, 4 L)before being absorbed onto silica gel (SiO₂, 1.5 Kg). The crude product,which was absorbed on silica gel, was purified by flash columnchromatography (SiO₂, 3.5 Kg, 0-25% EtOAc/hexanes gradient elution) toafford tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate (2, 414.7 g,679.8 g theoretical, 61% yield) as a white solid. For 2: ¹H NMR (300MHz, CDCl₃) δ 5.40 (m, 1H), 4.70 (m, 2H), 4.61 (m, 2H), 1.46 (s, 9H)ppm; C₁₀H₁₄N₂O₂ (MW, 194.23), LCMS (EI) m/e 217 (M⁺+Na).

Example 5. Synthesis of4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole Example V

4-Iodopyrazole (20)

A flask equipped with a nitrogen inlet, an addition funnel, athermowell, and a mechanical stirrer was charged with pyrazole (1, 450g, 6.62 mol) and tetrahydrofuran (THF, 5 L) at ambient temperature. Themixture was then cooled to 10° C. and N-iodosuccinimide (NIS, 1490 g,6.62 mol, 1.0 equiv) was added to the mixture in portions as a solid atapproximately 10° C. The resulting reaction mixture was then stirred atambient temperature for 1 hour (longer reaction times may be necessarydepending on ambient temperature). The mixture was then filtered and theTHF was removed under reduced pressure. The residue was suspended inethyl acetate (6 L) and insoluble materials were filtered. The darkfiltrate was sequentially washed with saturated aqueous sodiumthiosulfate solution (2×3 L) (organic layer lightens to a pale yellow),water (2×3 L), and brine (2 L). The resulting organic layer was thendried over sodium sulfate, filtered, and concentrated under reducedpressure to afford 4-iodopyrazole (1138 g, 1284.1 g theoretical, 88.6%)as a white to pale yellow solid after being dried in a vacuum oven atapproximately 30° C. overnight. ¹H NMR (400 MHz, DMSO-d₆) δ 13.17 (bs,1H), 7.93 (bs, 1H), 7.55 (bs, 1H) ppm; C₃H₃IN₂ (MW, 193.97), LCMS (EI)m/e 195 (M⁺+H).

1-Trimethylsilyl-4-iodopyrazole (21)

To a flask equipped with a reflux condenser, a nitrogen inlet,mechanical stirrer, and a thermowell was charged 4-iodopyrazole (200 g,1.03 mol) and THF (2 L) at ambient temperature. To this solution wasadded triethylamine (TEA, 158 mL, 1.13 mol, 1.1 equiv) and the resultingsolution was cooled to 0° C. in an ice-brine bath. To this solution wasadded chlorotrimethylsilane (TMS-Cl, 137 mL, 1.08 mol, 1.05 equiv) withvigorous stirring allowing the temperature to reach 18° C. (The reactionbecomes very thick and difficult to stir, but becomes manageable afterover time). When the exothermic process had subsided, the cold bath wasremoved and the reaction was warmed to room temperature. The reactionwas followed by GC and was found to be deemed complete after about 1hour (sampling of reaction must be done out of air and diluted with drysolvent to prevent TMS hydrolysis). The reaction mixture was thendiluted with n-heptane (2 L) before being filtered under nitrogen. Thesolvent was removed from the filtrate under reduced pressure venting therotovap with nitrogen. The residual oil was diluted with n-heptane (1 L)and re-concentrated. If the solids formed upon adding the n-heptane, asecond filtration was necessary. The residue was then distilled underthe reduced pressure (70-90° C. at about 0.5 Torr) using a Kugelohr toafford 1-trimethylsilyl-4-iodopyrazole (263 g, 274.1 g theoretical, 96%)as a colorless oil. This material must be kept under nitrogen at alltimes since the TMS group rapidly hydrolyzes. Subsequently, it was foundthat 1-trimethylsilyl-4-iodopyrazole can be prepared by heating theiodopyrazole with 2 equivalents of hexamethyldisilazane for 1 hr.

4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1)

A flask equipped with a mechanical stirrer, a nitrogen inlet, anaddition funnel and a thermowell was charged with1-trimethylsilyl-4-iodopyrazole (225.1 g, 0.85 mol) and THF (2200 mL) atambient temperature. This mixture was cooled to approximately −6° C. inan ice/salt/brine bath before a solution of isopropyl magnesium chloridein THF (2 M solution in THF, 510 mL, 1.02 mol, 1.2 equiv) was added at arate such that the internal temperature did not exceed 0° C. The extentof metal/halogen exchange was monitored by GC and was found completeafter about 10 min. To the orange brown solution was then added2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(isopropylpinacolborate, 347 mL, 1.7 mol, 2.0 equiv) slowly at firstkeeping the temperature below 0° C. and then fairly rapidly after abouthalf of the compound was added allowing the temperature to reach 5° C.(the reaction becomes quite thick and then thins out slowly). Thereaction is then stirred at 0° C. for 10 min before being warmed toambient temperature over 1 h and stirred at ambient temperature for anadditional 1 h. The reaction mixture was cooled to approximately 6° C.and the saturated aqueous ammonium chloride solution (NH₄Cl, 2.2 L) wasadded with a temperature increase to 25° C. The mixture was stirred for5 minutes before being diluted with toluene (10 L). The layers wereseparated (a large amount of solid is present in the aqueous layer) andthe organic layer was sequentially washed with water (6×2.2 L) and brine(2×2.2 L) before being dried over sodium sulfate (Na₂SO₄). The dryingreagent, sodium sulfate (Na₂SO₄), was removed by filtration and thesolution was concentrated under reduced pressure. Residual toluene wasco-evaporated with n-heptane to afford4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1, 90.3 g,164.9 g theoretical, 54.8%) as a white solid. For 1: ¹H NMR (400 MHz,DMSO-d₆) δ 13.08 (bs, 1H), 7.94 (s, 1H), 7.62 (s, 1H), 1.23 (s, 12H)ppm; C₉H₁₅BN₂O₂ (MW, 194.04), LCMS (EI) m/e 195 (M⁺+H).

Example 6. Alternative Synthesis of4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

4-Bromopyrazole (22)

Pyrazole (19, 34.0 g, 0.5 mol) and NBS (89.0 g, 0.5 mol, 1.0 equiv) weresuspended in water (625 ml) at ambient temperature. The resultingsuspension was stirred at ambient temperature for overnight. Thereaction mixture was then extracted with EtOAc (2×100 mL). The combinedEtOAc extracts were washed with aqueous Na₂S₂O₃ and brine, dried overNa₂SO₄, and concentrated under reduced pressure to afford crude4-bromopyrazole (72.0 g, 73.5 g theoretical, 98% yield) as white solids(GC purity: >98%), which was directly used in the subsequent reactionwithout further purification.

4-Bromo-1-(ethoxyethyl)-1H-pyrazole (23)

To a solution of 4-bromopyrazole (70.0 g, 0.476 mol) in CH₂Cl₂ (600 mL)was added a solution of 3.1 M HCl in dioxane (4 mL) and ethyl vinylether (41 g, 0.569 mol, 1.2 equiv) at ambient temperature. The resultingreaction mixture was stirred at ambient temperature for 3 h. Thereaction was quenched with aqueous NaHCO₃ and the two layers wereseparated. The organic layer was washed with water, dried over Na₂SO₄,and concentrated under reduced pressure to dryness to afford4-bromo-1-(ethoxyethyl)-1H-pyrazole (113 g, 104.3 g theoretical, 97%yield) as an oil (GC purity: 89%), which was directly used in thesubsequent reaction without further purification.

1-(Ethoxyethyl)-4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-pyrazole(24)

To a 100 ml solution of iPrMgCl.LiCl (50 mmol, 1.8 equiv) in THF wasadded 4-bromo-1-(ethoxyethyl)-1H-pyrazole (6.15 g, 28 mmol) at ambienttemperature. The resulting reaction mixture was stirred at ambienttemperature for 12 h and then cooled to −20° C. Methoxy pinacolborate(10.6 g, 67 mmol, 2.4 equiv) was then added to the reaction mixture at−20° C. The resulting mixture was stirred at 0-10° C. for 1 h. AqueousNH₄Cl was added to quench the reaction. The mixture was then extractedwith petroleum ether (PE). The combined PE extracts were washed withsaturated NaHCO₃, dried over Na₂SO₄ and concentrated under reducedpressure. The crude product was crystallized in PE to afford1-(ethoxyethyl)-4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-pyrazole(24, 4.2 g, 7.45 g theoretical, 56.4% yield) as a white to off-whitesolid (GC purity: 99%). For 24: ¹H NMR (DMSO-d₆, 400 MHz) δ 8.09 (s,1H), 8.58 (s, 1H), 7.62 (s, 1H), 5.55 (q, 1H, J=6.1 Hz), 3.37 (dq, 1H,J=7.1, 9.6 Hz), 3.12 (dq, 1H, J=7.0, 9.7 Hz), 1.56 (d, 3H, J=6.0 Hz),1.24 (s, 12H), 1.00 (t, 3H, J=7.0 Hz) ppm; C₁₃H₂₃BN₂O₃ (MW, 266.14),LCMS (EI) m/e 267 (M⁺+H).

4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1)

To a mixture of 2,3-dimethylbutane-2,3-diol (25.0 kg, 211.6 mol) and1-(1-ethoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(24, 55.0 kg, 206.7 mol) in 1,2-dichloroethane (750 kg) was slowly addeda solution of HCl in MTBE (25.0 kg, 20-30% of HCl) at 0-5° C. Theresulting reaction mixture was then stirred at 10-20° C. for 3-5 hours.After the selective deprotection reaction was complete as monitored byHPLC (1: below 1%), the reaction mixture was degassed and refilled withnitrogen before being cooled to −15° C. The cooled reaction mixture wasthen added triethylamine (TEA, 30.0 kg, 296.5 mol) to adjust pH to 7-8.The mixture was then gradually warmed to ambient temperature beforebeing treated with water (150 kg). The two phases were separated and theorganic layer was washed with brine (60 kg) and dried over sodiumsulfate (Na₂SO₄). The drying reagent, sodium sulfate (Na₂SO₄), wasremoved by filtration and the resulting solution was concentrated underreduced pressure at 40-50° C. to a thick oil. The residue was warmed to60-70° C. and diluted with petroleum ether (100 kg) at the sametemperature. The resulting mixture was then gradually cooled to ambienttemperature and subsequently to −5° C. and stirred at the sametemperature for 3 hours. The solids was collected by centrifugation anddried at 50-60° C. under vacuum to afford the crude desired product (1,33.75 kg, 40.11 kg theoretical, 84.1%). The crude desired product wasthen suspended in 1,2-dichloroethane (30 kg) and the resulting mixturewas heated to reflux until a clear solution was formed. To the hotsolution was then added petroleum ether (150 kg) at the sametemperature. The resulting mixture was then gradually cooled to ambienttemperature and subsequently to −5° C. and stirred and the sametemperature for 3 hours. The solids were collected by centrifugation anddried under vacuum at 50-60° C. to afford4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1, 31.0 kg,40.11 kg theoretical, 77.3%) as an off-white solid, which is identicalin every comparable aspect to the material synthesized by the syntheticmethod as described above in Example 5.

Example 7. Synthesis of 4-Chloro-7H-[pyrrolo[2,3-d]pyrimidine

4,6-Dichloropyrimidine-5-carbaldehyde (26)

In a 5 L 4-neck flask equipped with a mechanical stirrer, an additionfunnel, a condenser, a thermocouple, and a N₂ sweep into an aqueous NaOHscrubbing solution, phosphorous oxychloride (POCl₃, 1 L, 10.572 mol,4.82 equiv) was charged and cooled in an ice/salt bath.N,N-Dimethylformamide (DMF, 320 mL, 4.138 mol, 1.85 equiv) was thenadded dropwise to the flask at 0±2° C. After addition of approximately100 mL of DMF over approximately 0.5 h, crystallization occurred and thereaction temperature was increased from 0 to 10° C. Addition was stoppedand the mixture was allowed to re-cool to approximately 2° C. Theremaining DMF was added over 2.5 h at below 8° C. The suspension becamevery thick making stirring difficult. When addition of DMF was complete,the mixture was stirred at 3-5° C. for 0.5 h. 4,6-Dihydroxypyrimidine(250 g, 2.232 mol) was added portion wise as a solid. After about onethird of 4,6-dihydroxypyrimidine was added, the reaction mixture becamemore mobile, and a slow exothermic phenomena occurred with the reactiontemperature increasing to approximately 12° C. over 0.5 h. The remaining4,6-dihydroxypyrimidine was added portion wise over 0.25 h with thereaction temperature increasing from 12 to 27° C. The reactiontemperature was maintained at 25-27° C. with intermittent cooling duringwhich time the yellow suspension became thinner, then thicker onceagain. After the exothermic phenomenon subsided in about 1 h, thereaction mixture was heated slowly. At about 55° C. the reaction mixturebecame extremely thick and the second mild exothermic phenomenon wasoccurred. The heating mantle was removed while the reaction temperaturecontinued to increase to about 63° C. and remained at this temperaturefor several minutes before dropping. Heating of the mixture was resumeduntil gentle reflux (about 100° C.) was attained. At about 95° C. asteady, fairly rapid evolution of HCl gas began and the reaction mixturegradually thinned and darkened. After about 0.5 h, a clear brownsolution developed with the reflux temperature slowly increasing to 115°C. over 1.25 h. After a total of 2.5 h at reflux, the reaction mixturewas cooled to ambient temperature and stirred overnight at ambienttemperature. Excess amount of POCl₃ (as much as possible) was removedunder reduced pressure (bath temperature 45-50° C.). The thick residualbrown oil was poured very slowly into cold H₂O (5 L) in a 20 Lseparation funnel, adding ice as needed to maintain the aqueous mixturenear room temperature. The aqueous mixture was extracted with EtOAc (2×3L followed by 1×2 L). The combined EtOAc extracts were washed with H₂O(2×2.5 L), saturated NaHCO₃ aqueous solution (1 L), brine (1 L), driedover Na₂SO₄, filtered, and concentrated under reduced pressure (bathtemperature at 35° C.) to afford the crude4,6-dichloropyrimidine-5-carbaldehyde (270 g, 395 g theoretical, 68.4%)as yellow-orange solids. A 20 g portion of this crude material waspurified by Kugelrohr distillation (oven temperature at 90-100° C., 225mTorr) to give 15.3 g of pure 4,6-dichloropyrimidine-5-carbaldehyde as awhite solid that turned yellow on standing at room temperature. ¹H NMR(300 MHz, CDCl₃) δ 10.46 (s, 1H), 8.89 (s, 1H) ppm.

4-Amino-6-chloropyrimidine-5-carbaldehyde (27)

A solution of 7 M NH₃ in MeOH (265 mL, 1.855 mol, 2.0 equiv) was addedover 1.25 h to a solution of 4,6-dichloropyrimidine-5-carbaldehyde(163.7 g, 0.9301 mol) in toluene (3 L) at ambient temperature. Thereaction temperature slowly increased from 20 to 26° C. and a yellowsuspension formed. Mild cooling was applied to maintain the reactiontemperature at below 26° C. The suspension was stirred at ambienttemperature for 3.5 h before the solids were collected by filtration.The solids were washed with EtOAc (1 L). The filtrate was concentratedunder reduced pressure, and the solids were triturated with toluene andn-heptane (2:1 v/v, 600 mL), filtered and dried to give 71.1 g of4-amino-6-chloropyrimidine-5-carbaldehyde as a yellow solid. Theoriginal solid filtered from the reaction mixture contained additionalamount of 4-amino-6-chloropyrimidine-5-carbaldehyde. The product wasextracted from the filtered solid by stirring in EtOAc (1.25 L) for 1.5h, filtering, then stirring in THF (750 mL) for 1 h and again filtering.Both EtOAc and THF filtrates were concentrated under reduced pressure,and the resulting solids were triturated with toluene and n-heptane (2:1v/v, 450 mL), filtered and dried to give an additional 44.1 g of4-amino-6-chloropyrimidine-5-carbaldehyde as a yellow solid. Thecombined yield of 4-amino-6-chloropyrimidine-5-carbaldehyde (115.2 g,146.5 g theoretical) was 78.6%. ¹HNMR (300 MHz, DMSO-d₆) δ 10.23 (s,1H), 8.71 (bs, 1H), 8.55 (bs, 1H), 8.39 (s, 1H) ppm; C₅H₄ClN₃O (MW,157.56), LCMS (EI) m/e 158 (M⁺+H).

6-Chloro-5-(2-methoxyvinyl)pyrimidin-4-ylamine (28)

A suspension of (methoxymethyl)triphenylphosphonium chloride (276.0 g,0.807 mol, 1.1 equiv) in THF (1.5 L) was cooled in an ice/salt bath to−2° C. and 1 M potassium tert-butoxide (KO^(t)Bu) in THF (807 mL, 0.807mol, 1.1 equiv) was added over 1.5 h at −2 to −3° C. The deep red-orangemixture was stirred at −2 to −3° C. for 1 h.4-Amino-6-chloropyrimidine-5-carbaldehyde (115.2 g, 0.7338 mol, 1.0equiv) was then added portion wise to the reaction mixture as a solidform using THF (200 mL) to rinse the container and funnel. During theaddition the reaction temperature increased from −3 to 13° C. and abrown color developed. When the reaction temperature dropped to 10° C.,the cooling bath was removed and the reaction mixture was allowed towarm to ambient temperature and stirred at ambient temperature for 42 h.The reaction mixture was cooled to −2° C. before being quenched by theslow addition of saturated NH₄Cl aqueous solution (750 mL). The mixturewas concentrated under reduced pressure to remove most of the THF. Theresidue was partitioned between EtOAc (3 L) and H₂O (1 L). The organicphase was filtered to remove insoluble material at the interface, thenextracted with 2 N HCl (4×250 mL) followed by 3 N HCl (2×250 mL). Thecombined HCl extracts were back-extracted with EtOAc (500 mL) thenfiltered through Celite to remove insoluble material. The filtrate wascooled in an ice/brine bath, adjusted to pH 8 with a 6 N aqueous NaOHsolution and extracted with EtOAc (3×1 L). The combined EtOAc extractswere washed with brine (1 L), dried over Na₂SO₄, stirred with charcoal(10 g) and silica gel (10 g) for 1 h. The mixture was filtered throughCelite, washing the Celite pad with EtOAc (1 L). The filtrate wasconcentrated, co-evaporating residual EtOAc with n-heptane (500 mL). Theresulting tan solid was pumped under high vacuum for 2 h to afford crude6-chloro-5-(2-methoxyvinyl)pyrimidin-4-ylamine (72.3 g, 136.2 gtheoretical, 53.1%). The crude desired product was used in the followingreaction without further purification. A sample of crude product (2.3 g)was purified by silica gel column chromatography on, eluting with 0-35%EtOAc/n-heptane to give 1.7 g of pure6-chloro-5-(2-methoxyvinyl)pyrimidin-4-ylamine as a white solid, whichwas found to be a 1 to 2 mixture of E/Z isomers. ¹H NMR (300 MHz,DMSO-d₆) for E-isomer: δ 8.02 (s, 1H), 7.08 (bs, 2H), 6.92 (d, 1H,J=13.1), 5.35 (d, 1H, J=13.0 Hz), 3.68 (s, 3H) ppm and for Z-isomer: δ8.06 (s, 1H), 7.08 (bs, 2H), 6.37 (d, 1H, J=6.8 Hz), 5.02 (d, 1H, J=6.7Hz), 3.69 (s, 3H) ppm; C₇H₈ClN₃O (MW, 185.61), LCMS (EI) m/e 186/188(M⁺+H).

4-Chloro-7H-[pyrrolo[2,3-d]pyrimidine (4)

Concentrated HCl (5 mL) was added to a solution of crude6-chloro-5-(2-methoxyvinyl)pyrimidin-4-ylamine (70.0 g, 0.3784 mol) inTHF (700 mL) and the resulting reaction mixture was heated to reflux for7.5 h. On warming a light suspension was formed that graduallyre-dissolved. When the reaction was deemed complete as monitored byHPLC, the reaction mixture was cooled to ambient temperature and stirredat ambient temperature for overnight. Solid NaHCO₃ (15 g) was added tothe reaction mixture and the resulting mixture was stirred at ambienttemperature for 1 h. Charcoal (7 g), silica gel (7 g) and Na₂SO₄ (20 g)were added and the mixture was heated to 40° C. for 1 h. The mixture wasthen cooled to ambient temperature and filtered through Celite, washingthe Celite pad with THF (1 L). The filtrate was concentrated underreduced pressure and the resulting solid was dried under reducedpressure to afford crude 4-chloro-7H-[pyrrolo[2,3-d]pyrimidine (4, 58.1g, 58.1 g theoretical, 100%) as a yellow-brown solid. This crude desiredproduct was dissolved in EtOAc (1 L) at 50-55° C. and treated withactivated charcoal (3 g). The mixture was filtered while warm throughCelite and the Celite pad was washed with warm EtOAc (250 mL). Thefiltrate was concentrated to about 500 mL and the suspension was allowedto stand at ambient temperature for overnight. The suspension wassubsequently cooled to 0-5° C. for 2 h before the solids were collectedby filtration. The solids were dried to afford pure4-chloro-7H-[pyrrolo[2,3-d]pyrimidine (4, 54.5 g, 58.1 g theoretical,94%) as yellow-brown crystals. ¹H NMR (400 MHz, DMSO-d₆) δ 12.58 (bs,1H), 8.58 (s, 1H), 7.69 (d, 1H, J=3.5 Hz), 6.59 (d, 1H, J=3.5 Hz) ppm;LCMS (EI) m/e 154/156 (M⁺+H).

Example A: In Vitro JAK Kinase Assay

The compound of Formula I was tested for inhibitory activity of JAKtargets according to the following in vitro assay described in Park etal., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142) and JAK2 (a.a. 828-1132) with an N-terminalHis tag were expressed using baculovirus in insect cells and purified.The catalytic activity of JAK1 and JAK2 was assayed by measuring thephosphorylation of a biotinylated peptide. The phosphorylated peptidewas detected by homogenous time resolved fluorescence (HTRF). IC₅₀s ofcompounds were measured for each kinase in the 40 microL reactions thatcontain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) bufferwith 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC₅₀measurements, ATP concentration in the reactions was 1 mM. Reactionswere carried out at room temperature for 1 hr and then stopped with 20μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (PerkinElmer, Boston, Mass.). Binding to the Europium labeled antibody tookplace for 40 minutes and HTRF signal was measured on a Fusion platereader (Perkin Elmer, Boston, Mass.). The compound of Formula I and theadipic acid salt had an IC₅₀ at JAK1 of ≦5 nM (measured at 1 mM ATP)with a JAK2/JAK1 ratio of >10 (measured at 1 mM ATP).

Example B: Cellular Assays

Cancer cell lines dependent on cytokines and hence JAK/STAT signaltransduction, for growth, can be plated at 6000 cells per well (96 wellplate format) in RPMI 1640, 10% FBS, and 1 nG/mL of appropriatecytokine. Compounds can be added to the cells in DMSO/media (finalconcentration 0.2% DMSO) and incubated for 72 hours at 37° C., 5% CO₂.The effect of compound on cell viability is assessed using theCellTiter-Glo Luminescent Cell Viability Assay (Promega) followed byTopCount (Perkin Elmer, Boston, Mass.) quantitation. Potentialoff-target effects of compounds are measured in parallel using a non-JAKdriven cell line with the same assay readout. All experiments aretypically performed in duplicate.

The above cell lines can also be used to examine the effects ofcompounds on phosphorylation of JAK kinases or potential downstreamsubstrates such as STAT proteins, Akt, Shp2, or Erk. These experimentscan be performed following an overnight cytokine starvation, followed bya brief preincubation with compound (2 hours or less) and cytokinestimulation of approximately 1 hour or less. Proteins are then extractedfrom cells and analyzed by techniques familiar to those schooled in theart including Western blotting or ELISAs using antibodies that candifferentiate between phosphorylated and total protein. Theseexperiments can utilize normal or cancer cells to investigate theactivity of compounds on tumor cell survival biology or on mediators ofinflammatory disease. For example, with regards to the latter, cytokinessuch as IL-6, IL-12, IL-23, or IFN can be used to stimulate JAKactivation resulting in phosphorylation of STAT protein(s) andpotentially in transcriptional profiles (assessed by array or qPCRtechnology) or production and/or secretion of proteins, such as IL-17.The ability of compounds to inhibit these cytokine mediated effects canbe measured using techniques common to those schooled in the art.

Compounds herein can also be tested in cellular models designed toevaluate their potency and activity against mutant JAKs, for example,the JAK2V617F mutation found in myeloid proliferative disorders. Theseexperiments often utilize cytokine dependent cells of hematologicallineage (e.g. BaF/3) into which the wild-type or mutant JAK kinases areectopically expressed (James, C., et al. Nature 434:1144-1148; Staerk,J., et al. JBC 280:41893-41899). Endpoints include the effects ofcompounds on cell survival, proliferation, and phosphorylated JAK, STAT,Akt, or Erk proteins.

Certain compounds herein can be evaluated for their activity inhibitingT-cell proliferation. Such as assay can be considered a second cytokine(i.e. JAK) driven proliferation assay and also a simplistic assay ofimmune suppression or inhibition of immune activation. The following isa brief outline of how such experiments can be performed. Peripheralblood mononuclear cells (PBMCs) are prepared from human whole bloodsamples using Ficoll Hypaque separation method and T-cells (fraction2000) can be obtained from PBMCs by elutriation. Freshly isolated humanT-cells can be maintained in culture medium (RPMI 1640 supplemented with10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin) ata density of 2×10⁶ cells/ml at 37° C. for up to 2 days. For IL-2stimulated cell proliferation analysis, T-cells are first treated withPhytohemagglutinin (PHA) at a final concentration of 10 μg/mL for 72 h.After washing once with PBS, 6000 cells/well are plated in 96-wellplates and treated with compounds at different concentrations in theculture medium in the presence of 100 U/mL human IL-2 (ProSpec-TanyTechnoGene; Rehovot, Israel). The plates are incubated at 37° C. for 72h and the proliferation index is assessed using CellTiter-GloLuminescent reagents following the manufactory suggested protocol(Promega; Madison, Wis.).

Example C: In Vivo Anti-Tumor Efficacy

Compounds herein can be evaluated in human tumor xenograft models inimmune compromised mice. For example, a tumorigenic variant of the INA-6plasmacytoma cell line can be used to inoculate SCID mice subcutaneously(Burger, R., et al. Hematol J. 2:42-53, 2001). Tumor bearing animals canthen be randomized into drug or vehicle treatment groups and differentdoses of compounds can be administered by any number of the usual routesincluding oral, i.p., or continuous infusion using implantable pumps.Tumor growth is followed over time using calipers. Further, tumorsamples can be harvested at any time after the initiation of treatmentfor analysis as described above (Example B) to evaluate compound effectson JAK activity and downstream signaling pathways. In addition,selectivity of the compound(s) can be assessed using xenograft tumormodels that are driven by other know kinases (e.g. Bcr-Abl) such as theK562 tumor model.

Example D: Murine Skin Contact Delayed Hypersensitivity Response Test

Compounds herein can also be tested for their efficacies (of inhibitingJAK targets) in the T-cell driven murine delayed hypersensitivity testmodel. The murine skin contact delayed-type hypersensitivity (DTH)response is considered to be a valid model of clinical contactdermatitis, and other T-lymphocyte mediated immune disorders of theskin, such as psoriasis (Immunol Today. 1998 January; 19(1):37-44).Murine DTH shares multiple characteristics with psoriasis, including theimmune infiltrate, the accompanying increase in inflammatory cytokines,and keratinocyte hyperproliferation. Furthermore, many classes of agentsthat are efficacious in treating psoriasis in the clinic are alsoeffective inhibitors of the DTH response in mice (Agents Actions. 1993January; 38(1-2):116-21).

On Day 0 and 1, Balb/c mice are sensitized with a topical application,to their shaved abdomen with the antigen 2,4,dinitro-fluorobenzene(DNFB). On day 5, ears are measured for thickness using an engineer'smicrometer. This measurement is recorded and used as a baseline. Both ofthe animals' ears are then challenged by a topical application of DNFBin a total of 20 μL (10 μL on the internal pinna and 10 μL on theexternal pinna) at a concentration of 0.2%. Twenty-four to seventy-twohours after the challenge, ears are measured again. Treatment with thetest compounds is given throughout the sensitization and challengephases (day −1 to day 7) or prior to and throughout the challenge phase(usually afternoon of day 4 to day 7). Treatment of the test compounds(in different concentration) is administered either systemically ortopically (topical application of the treatment to the ears). Efficaciesof the test compounds are indicated by a reduction in ear swellingcomparing to the situation without the treatment. Compounds causing areduction of 20% or more were considered efficacious. In someexperiments, the mice are challenged but not sensitized (negativecontrol).

The inhibitive effect (inhibiting activation of the JAK-STAT pathways)of the test compounds can be confirmed by immunohistochemical analysis.Activation of the JAK-STAT pathway(s) results in the formation andtranslocation of functional transcription factors. Further, the influxof immune cells and the increased proliferation of keratinocytes shouldalso provide unique expression profile changes in the ear that can beinvestigated and quantified. Formalin fixed and paraffin embedded earsections (harvested after the challenge phase in the DTH model) aresubjected to immunohistochemical analysis using an antibody thatspecifically interacts with phosphorylated STAT3 (clone 58E12, CellSignaling Technologies). The mouse ears are treated with test compounds,vehicle, or dexamethasone (a clinically efficacious treatment forpsoriasis), or without any treatment, in the DTH model for comparisons.Test compounds and the dexamethasone can produce similar transcriptionalchanges both qualitatively and quantitatively, and both the testcompounds and dexamethasone can reduce the number of infiltrating cells.Both systemically and topical administration of the test compounds canproduce inhibitive effects, i.e., reduction in the number ofinfiltrating cells and inhibition of the transcriptional changes.

Example E: In Vivo Anti-Inflammatory Activity

Compounds herein can be evaluated in rodent or non-rodent modelsdesigned to replicate a single or complex inflammation response. Forinstance, rodent models of arthritis can be used to evaluate thetherapeutic potential of compounds dosed preventatively ortherapeutically. These models include but are not limited to mouse orrat collagen-induced arthritis, rat adjuvant-induced arthritis, andcollagen antibody-induced arthritis. Autoimmune diseases including, butnot limited to, multiple sclerosis, type I-diabetes mellitus,uveoretinitis, thyroditis, myasthenia gravis, immunoglobulinnephropathies, myocarditis, airway sensitization (asthma), lupus, orcolitis may also be used to evaluate the therapeutic potential ofcompounds herein. These models are well established in the researchcommunity and are familiar to those schooled in the art (CurrentProtocols in Immunology, Vol 3., Coligan, J. E. et al, Wiley Press.;Methods in Molecular Biology: Vol. 225, Inflammation Protocols.,Winyard, P. G. and Willoughby, D. A., Humana Press, 2003.).

Example F: Animal Models for the Treatment of Dry Eye, Uveitis, andConjunctivitis

Agents may be evaluated in one or more preclinical models of dry eyeknown to those schooled in the art including, but not limited to, therabbit concanavalin A (ConA) lacrimal gland model, the scopolamine mousemodel (subcutaneous or transdermal), the Botulinumn mouse lacrimal glandmodel, or any of a number of spontaneous rodent autoimmune models thatresult in ocular gland dysfunction (e.g. NOD-SCID, MRL/lpr, or NZB/NZW)(Barabino et al., Experimental Eye Research 2004, 79, 613-621 andSchrader et al., Developmental Opthalmology, Karger 2008, 41, 298-312,each of which is incorporated herein by reference in its entirety).Endpoints in these models may include histopathology of the ocularglands and eye (cornea, etc.) and possibly the classic Schirmer test ormodified versions thereof (Barabino et al.) which measure tearproduction. Activity may be assessed by dosing via multiple routes ofadministration (e.g. systemic or topical) which may begin prior to orafter measurable disease exists.

Agents may be evaluated in one or more preclinical models of uveitisknown to those schooled in the art. These include, but are not limitedto, models of experimental autoimmune uveitis (EAU) and endotoxininduced uveitis (EIU). EAU experiments may be performed in the rabbit,rat, or mouse and may involve passive or activate immunization. Forinstance, any of a number or retinal antigens may be used to sensitizeanimals to a relevant immunogen after which animals may be challengedocuarly with the same antigen. The EIU model is more acute and involveslocal or systemic administration of lipopolysaccaride at sublethaldoses. Endpoints for both the EIU and EAU models may include fundoscopicexam, histopathology amongst others. These models are reviewed by Smithet al. (Immunology and Cell Biology 1998, 76, 497-512, which isincorporated herein by reference in its entirety). Activity is assessedby dosing via multiple routes of administration (e.g. systemic ortopical) which may begin prior to or after measurable disease exists.Some models listed above may also develop scleritis/episcleritis,chorioditis, cyclitis, or iritis and are therefore useful ininvestigating the potential activity of compounds for the therapeutictreatment of these diseases.

Agents may also be evaluated in one or more preclinical models ofconjunctivitis known those schooled in the art. These include, but arenot limited to, rodent models utilizing guinea-pig, rat, or mouse. Theguinea-pig models include those utilizing active or passive immunizationand/or immune challenge protocols with antigens such as ovalbumin orragweed (reviewed in Groneberg, D. A., et al., Allergy 2003, 58,1101-1113, which is incorporated herein by reference in its entirety).Rat and mouse models are similar in general design to those in theguinea-pig (also reviewed by Groneberg). Activity may be assessed bydosing via multiple routes of administration (e.g. systemic or topical)which may begin prior to or after measurable disease exists. Endpointsfor such studies may include, for example, histological, immunological,biochemical, or molecular analysis of ocular tissues such as theconjunctiva.

Example G: In Vivo Protection of Bone

Compounds may be evaluated in various preclinical models of osteopenia,osteoporosis, or bone resorption known to those schooled in the art. Forexample, ovariectomized rodents may be used to evaluate the ability ofcompounds to affect signs and markers of bone remodeling and/or density(W. S. S. Jee and W. Yao, J Musculoskel. Nueron. Interact., 2001, 1(3),193-207, which is incorporated herein by reference in its entirety).Alternatively, bone density and architecture may be evaluated in controlor compound treated rodents in models of therapy (e.g. glucocorticoid)induced osteopenia (Yao, et al. Arthritis and Rheumatism, 2008, 58(6),3485-3497; and id. 58(11), 1674-1686, both of which are incorporatedherein by reference in its entirety). In addition, the effects ofcompounds on bone resorption and density may be evaluable in the rodentmodels of arthritis discussed above (Example E). Endpoints for all thesemodels may vary but often include histological and radiologicalassessments as well as immunohisotology and appropriate biochemicalmarkers of bone remodeling.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A process of preparing a compound of Formula I:

or a salt thereof, comprising: reacting a compound of Formula IIIa:

with a compound of Formula IVa:

under Suzuki coupling conditions to form a compound of Formula IIa:

deprotecting the compound of Formula IIa by reacting with an inorganicacid to form a compound of Formula V:

and reacting the compound of Formula V with a compound of Formula VI:

in the presence of a reducing agent to form a compound of Formula I, ora salt thereof.
 2. The process according to claim 1, wherein the Suzukicoupling conditions comprise heating a reaction mixture comprising thecompound of Formula IIIa, the compound of Formula IVa, a Suzuki couplingcatalyst, a base and a solvent component.
 3. The process according toclaim 2, wherein the Suzuki coupling catalyst is Pd(dppf)₂Cl₂,[1,1′-bis(dicyclohexylphosphino)ferrocene]dichloropalladium (II),tetrakis(triphenylphosphine)palladium(0), ortetrakis(tri(o-tolyl)phosphine)palladium(0).
 4. The process according toclaim 2, wherein the Suzuki coupling catalyst is[1,1′-bis(dicyclohexylphosphino)ferrocene]dichloropalladium (II).
 5. Theprocess according to claim 2, wherein the base is sodium carbonate,potassium carbonate, or cesium fluoride.
 6. The process according toclaim 2, wherein the base is cesium fluoride.
 7. The process accordingto claim 6, wherein the cesium fluoride is present in 3 equivalents ormore based on the compound of Formula IVa.
 8. The process according toclaim 2, wherein the solvent component comprises tert-butanol and water.9. The process according to claim 1, wherein the compounds of FormulaIIIa and IVa are present in about a 1:1 molar ratio.
 10. The processaccording to claim 1, wherein the inorganic acid is present in an amountof 5 to 8 equivalents based on the compound of Formula IIa, and whereinthe inorganic acid is hydrochloric acid.
 11. The process according toclaim 1, wherein the inorganic acid is hydrochloric acid.
 12. Theprocess according to claim 1, wherein said reacting of the compound ofFormula V with the compound of Formula VI is carried out in the presenceof at least two equivalents of a second base.
 13. The process accordingto claim 12, wherein the second base is a tertiary amine.
 14. Theprocess according to claim 12, wherein the second base is triethylamine.15. The process according to claim 1, wherein the reducing agent issodium cyanoborohydride or sodium triacetoxyborohydride.
 16. The processaccording to claim 1, wherein the reducing agent is sodiumtriacetoxyborohydride.
 17. The process according to claim 16, whereingreater than 1 equivalent of sodium triacetoxyborohydride is used basedon the compound of Formula V.
 18. The process according to claim 1,wherein greater than 1 equivalent of the compound of Formula VI is usedbased on the compound of Formula V.
 19. The process according to claim1, wherein the reacting of the compound of Formula V with the compoundof Formula VI is performed in a halogenated solvent.
 20. The processaccording to claim 19, wherein the halogenated solvent isdichloromethane.
 21. The process according to claim 1, furthercomprising reacting the compound of Formula I with adipic acid to formthe adipate salt of the compound of Formula I.